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Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): from Assay Set-up to Data Acquisition
细胞因子流式细胞检测法量化恒河猴T细胞特异性抗原的记忆反应:从分析计划到数据采集   

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Abstract

What was initially termed ‘CFC’ (Cytokine Flow Cytometry’) is now more commonly known as ‘ICS’ (Intra Cellular Staining), or less commonly as ‘ICCS’ (Intra Cellular Cytokine Staining). The key innovations were use of an effective permeant (allowing intracellular staining), and a reagent to disrupt secretion (trapping cytokines, thereby enabling accumulation of detectable intracellular signal). Because not all researchers who use the technique are interested in cytokines, the ‘ICS’ term has gained favor, though ‘CFC’ will be used here.
CFC is a test of cell function, exposing lymphocytes to antigen in culture, then measuring any cytokine responses elicited. Test cultures are processed so as to stain cells with monoclonal antibodies tagged with fluorescent markers, and to chemically fix the cells and decontaminate the samples, using paraformaldehyde.
CFC provides the powers of flow cytometry, which includes bulk sampling and multi-parametric cross-correlation, to the analysis of antigen-specific memory responses. A researcher using CFC is able to phenotypically characterize cells cultured with test antigen, and for phenotypic subsets (e.g. CD4+ or CD8+ T cells) determine the % frequency producing cytokine above background level.
In contrast to ELISPOT and Luminex methods, CFC can correlate production of multiple cytokines from particular, phenotypically-characterized cells. The CFC assay is useful for detecting that an individual has had an antigen exposure (as in population screenings), or for following the emergence and persistence of antigen memories (as in studies of vaccination, infections, or pathogenesis). In addition to quantifying the % frequency of antigen-responding cells, mean fluorescence intensity can be used to assess how much of a cytokine is generated within responding cells.
With the technological advance of flow cytometry, a current user of CFC often has access to 11 fluorescent channels (or even 18), making it possible to either highly-characterize the phenotypes of antigen-responding cells, or else simultaneously quantify the responses according to many cytokines or activation markers. Powerful software like FlowJo (TreeStar) and SPICE (NIAID) can be used to analyse the data, and to do sophisticated multivariate analysis of cytokine responses.
The method described here is customized for cells from Rhesus macaque monkeys, and the extensive annotating notes represent a decade of accumulated technical experience. The same scheme is readily applicable to other mammalian cells (e.g. human or mouse), though the exact antibody clones will differ according to host system. The basic method described here incubates 1 x 106 Lymphocytes in 1 ml tube culture with antigen and co-stimulatory antibodies in the presence of Brefeldin A, prior to staining and fixation.

Keywords: ICS(ICS), CFC(CFC), Cytometry(流式细胞仪), ICCS(ICCS), Antigen(抗原)

[Historical Background] The first report of fixing and permeabilizing lymphocytes, then staining them with antibodies against IFN gamma, was made by Andersson et al. in 1989. In 1991, Sander et al. demonstrated improved methods, using paraformaldehyde to fix cells, saponin (an amphipathic glycoside) to permeabilize them, and fluorescently-labeled antibodies to stain intracellular cytokines for microscope examination. In 1993, Jung et al. extended this method for use with flow cytometry, included monensin (a polyether antibiotic ionophore which blocks intracellular protein transport) to inhibit secretion, so as to increase the intracellular signal of the cytokine molecules that would otherwise be released soon after synthesis. In 1995, Prussin and Metcalfe used directly-conjugated antibodies, and reported good results with 6 h incubations. Also in 1995, Picker et al. considerably enhanced the sensitivity and reproducibility of cytokine detection by using Brefeldin A (‘BfA’, a fungal lactone antibiotic) to block the cytokine-secretion apparatus, and by using a different permeant (Tween-20). This improved method was applied by Picker et al. in a 1997 report of the antigen-specific homeostatic mechanism in human HIV+ patients. In 2001, Schuerwegh et al. confirmed that BfA provides better cytokine signal in the assay than does monensin, though monensin is still used widely by others in this method.
Regarding non-human primate studies, two reports in 1989, one by Gardner and another by McClure, showed that Rhesus macaques were a useful model for studying HIV disease and AIDS. In 2002, Picker et al. reported the application of a specially-modified CFC assay to Rhesus macaques. In 2012, a consortium-appointed group aiming to establish standards for collaborating groups using CFC in Rhesus vaccine studies published their recommendations for a 96-well plate method with a 6 h total incubation (Donaldson et al., 2012; Foulds et al., 2012).
The general procedure reported here is that 2002 tube-format (see Note 1) method, now with a 9 h total incubation, and optimized especially for low-end sensitivity. The specific details here are the state of the art now practiced by the Picker Lab, at the Oregon Health and Science University, affiliated with the Oregon National Primate Research Center. These methods have been used in several of our recent publications (Hansen et al., 2013a; Hansen et al., 2013b, Fukazawa et al., 2012; Hansen et al., 2011; Hansen et al., 2009). It is important to note that in our hands, plate-format CFC is not as sensitive and reproducible for weak responses as is this tube-based method described here (unpublished observations). Until that difference is understood and solved, the tube-based method remains the most-sensitive format for CFC.

Materials and Reagents

  1. Lymphocyte suspension from Rhesus macaques blood (Note 2) or bronchoaveolar lavage (BAL), or harvest from solid biopsy or necropsy tissue, cell density determined by a method accurate for the sample (Notes 3 and 4)
    1. Need ~1 x 106 viable lymphocytes per test
    2. Freshly-obtained (Note 5)
    3. OR thawed cryopreserved sample (Note 6)
  2. Antigen
    1. Negative control(s) (Note 9)
    2. Positive control:
      Superantigen Staphylococcus Enterotoxin B SEB (Note 10) (Toxin Technology, catalog number: BT202 ) (lyophilized powder, 100 μg; stock: 100 μg/ml in water; usage: 2 μl/test)
    3. Other positive control (experiment-specific)
    4. Peptide mixes (1-100 different peptides, at ≥ 2 μg/peptide/1 ml-test) 15 amino acid peptides (15 mers) overlapping by 11 amino acids 
  3. Antibody
    1. Unconjugated antibody for costimulation during culture incubation (Note 11)
      Anti-CD28, pure unconjugated, clone CD29.2 (Note 12)
      Anti-CD49d, pure unconjugated, clone 9F10
      Stocks diluted to 0.5 mg/ml; use 1 μl per 1 x 106 Ly
    2. Essential fluorophore-conjugated monoclonal antibodies
      The fluorophores you use are dependent upon the flow cytometer available to you. Many companies sell the appropriate fluorophore-conjugated antibodies, including BD, Beckman Coulter, Life Technologies, InvitrogenTM, eBiosciences, and many others.
      Anti-CD3e, clones reactive with Rhesus (SP34-2, FN18)
      Anti-CD4 (L200, MT477))
      Anti-CD8a (SK1, RPA-T8)
      Anti-CD69 (FN50, CH/4, TP1.55.3) (Note 13)
      Anti-IFNg (B27)
      Anti-TNFa (MAB11)
    3. Optional fluorophore-conjugated monoclonal antibodies
      Anti-CD45 (DO58-1283) (Note 14)
      Anti-IL2 (MQ1-17H12)
      Anti-MIP1b (D21-1351)
      Ant-CD107 (alpha: H4A3, beta: H4B4) (Note 15)
      Anti-CD95 (DX2)
      Anti- CD45RA (L48, 5H9, MEM-56, others) (Note 41)
      Anti-CCR7 (CD197) (Note 42)
      Anti-Ki67 (B56) (Note 38)
  4. Brefeldin A (Sigma-Aldrich, catalog number: B-7651 )
    Vendors:  (Sigma-Aldrich, catalog number: B-7651; BioLegend, catalog number: 91850 )
    Working stock: 10 mg/ml, in DMSO (1.0 μl/test) (Note 16)
  5. Benzonase (Merck KgaA, Novagen) (use at 50 U/ml)
  6. 1x RPMI-1640 (w/o L-glutamine, 0.1 μm filtered) (e.g. HyCone, catalog number: SH30096.02 )
  7. Fetal Bovine Serum (FBS, aka: 'FCS') (e.g. HyClone, catalog number: SH30070.03 ) (defined, heat-inactivated, 40 nm-filtered)
  8. Penicillin+Streptomycin (P/S) Solution (e.g. Sigma-Aldrich, catalog number: P-0781 )
  9. L-glutamine (200 mM) (e.g. Sigma-Aldrich, catalog number: G-7513 (100 ml)
  10. Sodium pyruvate (SP) (e.g. Sigma-Aldrich, catalog number: S-8636 ) (100 ml)
  11. Beta-Mercaptoethanol (bME) (e.g. Sigma-Aldrich, catalog number: M-7522 ) (100 ml) (Note 40)
  12. Sterile-filtration apparatus (e.g. Corning, catalog number: 430769 ) (500 ml capacity 0.22 μm cellulose-acetate filter)
  13. Dulbecco's Phosphate Buffered Saline (DPBS) (e.g. Thermo Fisher Scientific, Corning, catalog number: 55-031-PB )
  14. Bovine serum albumin (BSA) (e.g. Thermo Fisher Scientific, catalog number: BP1605100 )
  15. Sodium azide (preservative; NaN3) (e.g. Thermo Fisher Scientific, catalog number: BP922-500 )
  16. BD FACS Lysing Solution (10x concentrate) (BD, catalog number: 349202 )
  17. Tween-20 (polyoxyethylenesorbitan monolaurate) (e.g. Sigma-Aldrich, catalog number: P-7949 )
  18. Aqua LIVE/DEAD kit (Life Technologies, InvitrogenTM, www.lifetechnologies.com, search 'LIVE/DEAD' for an evolving array of stains and kits) (Note 43)
    1. Concentrated dye stock (see Recipes)
    2. Staining Solution (made fresh) (see Recipes)
  19. Tissue culture medium ('R10') (see Recipes)
  20. 'PAB' wash buffer (see Recipes)
  21. 'Lyse' fixation and RBC-lysing solution (see Recipes) (Note 17)
  22. 'Perm' fixation and cell-permeabilizing solution (see Recipes) (Note 18)

Equipment

  1. Tubes (Notes 1 and 7) polypropylene (PP) (round-bottom, 5 ml/12 x 75 mm, sterile) (e.g. Falcon®, catalog number: 35-2054 )
  2. Computer-generated printed labels for tubes (optional; must stick well to polypropylene)
  3. Tube-holding racks (optional) (e.g. Thermo Fisher Scientific, No-Wire Grip Rack 10-13 MM 90 place) (Note 8)
  4. Foam cosmetic wedges (or functional equivalent) (The ones we use are 2" long x ¾" high when lying on the long side.)
  5. Laminar flow biosafety cabinet (for sterility, even if not working with pathogens)
  6. Trapped vacuum aspirator
  7. Appropriate fluid measuring dispensers, with appropriate disposables
    1. Electric pump pipettors for measurements between 1-50 ml
    2. Manual hand micropipettors for measurements between 0.5-1,000 μl
    3. Repeater-dispensers (e.g. from Eppendorf)
    4. Hand repeaters, for measurements between 0.5-2 ml
    5. Stationary pump dispensers, for measurements between 1-5 ml
  8. Centrifuge with swing-buckets (capable of 800 x g) (e.g. Sorvall Legend T/RT)
  9. Vortexer
  10. Lab timer
  11. Incubator for tissue culture (humidified, stable at 37 °C, 5% CO2 atmosphere)
    Option: Standard water-jacketed T/C incubator (e.g. Thermo Fisher Scientific, FormaTM, Series II, Model: 3110 )
    Option: UniBator (Tritech Research) DigiTherm CO2 incubator with rapid cooling and bi-directional interface (Note 19)
  12. Refrigerator at 4 °C
  13. Flow cytometry analyser, 6-fluorescence detectors or more (e.g. BD, model: LSR-II )
  14. A method of counting PBMC (e.g., Coulter counter, Guava, or hemocytometer)

Procedure

Note: Please see Figures 1 and 2 for the flow chart of the CFC set up process and the CFC tube staining process, respectively.


Figure 1. Flow chart of the CFC set up process


Figure 2. Flow chart of the CFC tube staining process

Part I. Culture setup, incubation, chilling

  1. Pre-start preparation
    1. See QA/QC notes.
    2. Make fresh medium (or begin warming to room temperature).
    3. Label PP tubes.
    4. Begin thawing peptides in DMSO (if applicable).
  2. Obtain cells
    1. Freshly-harvested WBC (Notes 2-5).
    2. PBMC (peripheral blood mononuclear cells) from blood. (Other protocols explain how to isolate PBMC from blood. It is also possible to use whole blood that has been treated with a hypotonic saline like ACK to burst red blood cells.)
    3. BAL (bronchoaveolar lavage, sieved).
    4. Solid tissue, mechanically processed (LN, spleen, bone).
    5. Solid tissue, enzymatically processed (gut, liver, vagina).
    6. Cryopreserved WBC harvests.
      1. Thaw (Note 20).
      2. Rest (Note 20).
      3. Determine viability (Note 20).
  3. Adjust suspension density to approximately 2 x 106 Ly/ml, in fresh-made R10 (Note 21)
    1. e.g. 1 x 106 Ly/500 μ.
    2. R10 un-supplemented by antibodies or antige.
    3. Set suspension aside at room temperature until use.
  4. Prepare R10 supplemented with costimulating MAb anti-CD28, anti-CD49d (Note 11)
    1. Determine the total volume needed for the assay setup: (# tubes) x (500 μl/tube) x 1.2 (i.e., increase by 20%).
    2. Measure R10 into vessel labeled 'costim-R10'.
    3. Add anti-CD28 and anti-CD49d:
      1. Presuming [stock] = 0.5 mg/ml.
      2. Use 1 μl/500 μl (i.e., 2 μl/ml R10).
      3. Mix (vortex or inversion).
  5. Prepare substocks of R10 + costim, supplemented with antigens
    1. Determine the total volume needed for a particular antigen (# tubes) x (500 μl/tube) x 1.1 (i.e., increase by 10%).
    2. Measure R10 + costim into vessel labeled by antigen.
      1. Briefly vortex the stock antigen is mixed and homogenous.
      2. Ensure that the measurement is accurate (Note 22).
      3. Mix (vortex or inversion).
  6. Dispense 500 μl of R10 + costim + Ag into all appropriately-labeled tubes
    1. Use the tube labels to consistently orient the tubes in a reproducible way in the holding rack(s).
    2. Vortex the solution immediately before using (it is convenient to use a repeater for this dispensing).
    3. Dispense the 500 μl/tube, aiming down to a particular 'side' the tube (that side is now a possible cause of cross-contamination when using a repeater, so subsequent steps should orient the tubes differently, to avoid cross-contamination).
    4. Put the tube-holding rack(s) in a 37 °C, 5% CO2 incubator until use (these tubes can be prepared up to 2 h before adding cells).
  7. Dispense 500 μl of cell suspension into all racked tubes
    1. Be attentive to the label orientation on the tubes; orient the tubes such that dispensed suspension goes down an opposite side of the tubes (to minimize the risk of cross-contamination).
    2. It is convenient to use a repeater for this step.
    3. Always dispense into 'Negative' control tubes first.
    4. Always dispense into 'SEB' positive control tubes last.
    5. This step should take place as quickly as possible, since it is combining cells with antigen. At room temperature, however, the cells are not yet very metabolically active.
    6. It is not necessary to individually vortex tubes after this combination.
  8. Transfer the racks to an incubator stably at 37 °C, 5% CO2
    1. Ensure that a humidifying tray contains water (or else evaporation from the tubes will concentrate cells, Ag, and salts, changing the assay outcome).
    2. Place the rack on its side, resting the rack top on a cosmetic wedge. The sharp angling of the tubes makes the medium more shallow, making gas diffusion easier. This orientation also makes it so that the cells settle over a larger area, with less cell-cell contact. This seems to improve response sensitivity, for reasons that remain unclear.
    3. Set a timer for 1 h.


      Figure 3. How to angle a rack of ICS tubes during incubation

  9. After 1 h of incubation,add Brefeldin A to all tubes (Note 24)
    1. Prepare a dilute cocktail, to be used at 50 μl/tube.
      1. If (BfA stock) is 10 mg/ml, use 1 μl/tube.
      2. Dilute the BfA into unsupplemented R10. 1 part BfA into 39 parts R10; deliver 40 μl of this mix into each ICS tube. A 40 μl drop will reliably slide down the wall of a PE tube.
    2. Using a repeater, shoot the BfA downward into the tube.
      1. Orient the labels to minimize cross-contamination.
      2. Dispense in a way to minimize the disruption of the settled cell pellets in each tube.
      3. 40 μl produces a heavy enough drop toreliably run down the side of the tube.
    3. Do not vortex or further mix the tube contents.
    4. The tubes should be uncapped (or with loosely-applied 'snap caps'. It is important that easy gas exchange is allowed. The pen/strep is typically very effective at preventing bacterial and fungal growth during the short incubation period.
  10. Return the racks to the incubator (humidified, 37 °C, 5% CO2)
    1. Incubate tubes for an additional 5-8 h (with rack angled as in Figure 3) (Note 25).
  11. At the end of the incubation, the racks should be cooled
    1. The simplest way to accomplish this is to transfer the racks to a refrigerator at 4 °C.
    2. Tritech Research sells novel incubators that can, by computer-clock control, cool rapidly (37 °C to 15 °C, in less than 20 min; eventually to 4 °C) (Note 19).

Part II. Staining

  1. Samples should be processed to fixation within 12 h
    1. Although the mammalian cells are metabolically nearly inert at 4 °C, they remain alive. Left too long, the CFC signal will change.
    2. Transfer the culture tubes from the incubation racks to centrifuge bucket inserts.
  2. Wash
    1. Using a bottletop pump dispenser, fill all tubes with PAB wash buffer.
    2. Centrifuge 800 x g, 10 min (sufficient to produce a tight cell pellet in a 5 ml tube). The temperature of the run doesn't matter much, so long as it is well below metabolically-active 37 °C; the cells will soon be fixed.
    3. Remove supernatant (Note 26)
      1. Method 1: Wand vacuum-aspiration (Note 27).
      2. Method 2: Wand vacuum-aspiration with stop (Note 28).
      3. Method 3: Decanting (Note 29).
      4. Method 4: Decanting with insert gasket (Note 30).
    4. Resuspend pellets (Video 1) (Note 31).

      Video 1. Hand-decanting wash supernatant from polypropylene tubes used in CFC (ICS) assay

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

  3. Stain with Aqua LIVE/DEAD discriminator dye (optional, esp. for thawed cells)
    1. Wash the cells with ice-cold 1x PBS.
    2. Aspirate to remove supernatant (leaving minimal residual fluid).
    3. Resuspend cells (as in Part II, step B4) in 100 μl ice-cold 'Staining Solution' (100 μl/1 x 106 Ly).
      1. Incubate 20 min in dark, on ice.
    4. Wash (see Part II-B) to stop.
  4. Apply surface antibody cocktail (Note 32)
    1. Cocktail can be made hours ahead of time, in PAB.
    2. Formulate cocktail at 50 μl/tube Example: CD45, CD4 (Note 44).
    3. Dispense with a repeater.
    4. Vortex the tubes after adding the cocktail to the tubes.
    5. Incubate at room temperature for 30 min (in the dark) (Note 33).
    6. If the surface stain includes a two-stage biotin-streptavidin stain, the first cocktail should include the biotinylated reagent. After the 30 min incubation, add the streptavidin reagent to the tubes (no pre-washing is needed). It is convenient to put the streptavidin in a 50 μl/tube PAB dilution, which allows for repeater dispensing.
  5. Wash (see Part II-B)
  6. Apply 1 ml 'Lyse' per tube (Notes 17 and 34)
    1. Set a lab timer for 10 min.
    2. Ensure all the pellets are effectively disrupted.
    3. Have a plan figured out that will allow vortexing within two minutes of when you start to dispense Lyse, and that ensures the tubes get vortexed in the same order the Lyse was added.
    4. Fill an appropriately-sized repeater.
    5. Dispense quickly into tubes.
      1. Start the timer before adding to the first tube.
      2. Aim the repeater towards the midpoint wall.
    6. Begin vortexing tubes within 2 min of receiving Lyse.
      1. How Lyse is added is one of the most important details in this entire process. If the cells are not well-resuspended prior to adding Lyse, they will be fixed together, degrading data quality. If you do not mix the cells and Lyse quickly after combining them, you might get poor rupture of RBCs, degrading data quality. It is important to vortex soon and vigorously after adding the Lyse.
      2. If time allows, vortex the tubes again (this is especially valuable if the samples contain RBC contamination).
    7. Incubate ten full minutes at room temperature, in the dark.
  7. Wash (see Part II-B)
    Adding PAB reduces the hypotonicity of the Lyse reagent, slowing the osmotic swelling of the cells, and diluting the paraformaldehyde chemically fixing the cellular proteins. The residual fluids will match those dilutions (Note 35).
  8. Apply 0.5 ml 'Perm' per tube (Note 18)
    Basically, repeat the same method used for 'Lyse', but with 0.5 ml/tube.
  9. Wash (Note 36)
  10. Wash (Note 37)
  11. Apply intracellular antibody cocktail (Note 32)
    1. Cocktail can be made hours ahead of time, in PAB.
    2. Formulate cocktail at 50 μl/tube Example: CD3, CD8, CD69, cytokines (Note 44).
    3. Dispense with a repeater.
    4. Vortex the tubes after adding the cocktail to the tubes.
    5. Incubate at room temperature for 30-45 min (at room temperature, in the dark) (Note 33).
      If the cocktail includes Ki67, the incubation should be lengthened to 45 min (Note 38).
  12. Wash (Note 39)
    1. Remove as much fluid as possible, to ensure fast acquisition times.

Part III. Data Acquisition

  1. See Notes about QA/QC.
  2. Samples should be acquired within 72 h (Notes 46 and 47).

Notes

  1. Presented here is exclusively the tube-format method. While it is true that plate-based methods provide many advantages (multi-channel processing, smaller reagent volumes, streamlined process, robotic acquisition), we learned the hard way that some aspect of that method results in a loss of low-end sensitivity to test antigen. Whereas both the tube and plate method yield comparable CFC results above approximately 0.2% cytokine response, the tube method in our hands consistently performs better with weak responses below that level. We recognized this by 2007, and have been unable yet to modify a 96-well plate method to achieve the same low-end results.
  2. When using blood, an anticoagulant must be used, otherwise the cells of interest will be lost in clot formation. The Picker Lab prefers ACD (acid-citrate-dextrose, 'sodium citrate') as an anticoagulant. Repeated assessments have demonstrated (unpublished observtions) that CFC results are qualitatively different, and quantitatively less, when heparin is used. In our work, virus released to the extracellular fluid is quantified using PCR, so the anticoagulant chelating agent EDTA cannot be used. Otherwise, it has been demonstrated to work in CFC as well as ACD.
  3. Coulter counters (e.g. the Beckman Coulter Ac*T diff5 noted in the Equipment section) are designed to quantify human cells in blood, though they can be calibrated for Rhesus cells. The technology, however is not designed for samples containing significant non- or sub-cellular debris, as can be plentiful in cryopreserved samples recovered from a thaw, or from solid tissue biopsy mechanically and/or enzymatically treated to harvest lymphocytes. In any case not starting with fresh blood, a Coulter counter will report cell density inaccurately, usually by overcounting. Moreover, this kind of counter cannot reliably distinguish between living and dead cells. In our careful (unpublished) examinations of this issue, we found that these counters over-count lymphocyte harvests from lymph node and spleen, typically by a factor ranging from 1-2 fold. Lymphocyte harvests from lung wash or resulting from enzymatically-digested tissues (e.g. liver, gut) are functionally uncountable with a counter based on this technology because of wildly-variable amounts of debris that can populate the counting gates. And in cases of samples undergoing freeze-thaw, the viability cannot be assessed reliably. In these cases, other technologies must either augment the counts (e.g. a viability stain), or replace the Coulter counter technology. One widely-used alternative is a Guava counter, using one pass with a live-dead stain (e.g. ViaCount), and another pass gating by at least scatterplot and CD3+. This can be expensive and time-consuming. It doesn't matter exactly how one counts the cells for this assay, but users should be aware that most counting methods (even hemocytometers) are inaccurate for clinical non-blood WBC. Since all these samples will be collected on a flow cytometer, which is an instrument capable of reporting a precise number regarding the number of T cells assayed, the assay itself provides a means to QC the counting method used for input.
  4. The CFC method described in this protocol presumes an input of 1 x 106 lymphocytes per test, and antibody and reagent inputs are meant to be proportional. In practice, however, it has been difficult to correctly pre-quantify lymphocytes obtained from bronchoaveolar lavage (BAL) or from the hours-long enzymatic harvests from gut or liver tissue, so empirically-determined approximations are used. We have found (unpublished) that in 'dirty' cell suspension inputs (e.g. BAL, enzymatic digestions) that the strict proportionality of lymphocytes to lymphocyte-marking antibodies breaks down. For example, if a Coulter count reports that a BAL input into a CFC tube is 1e6 lymphocytes, but in actuality the input was 1/20 of that, we have found that using 1/20 of the staining antibodies yields qualitatively and quantitatively worse results than just presuming that the input was actually 1e6 lymphocytes. We hypothesize that the explanation is that 'dirty' samples have plentiful low-affinity non-specific binding sites, which 'sponge-up' much of the antibody reagent. Therefore, the CFC method described here still works effectively with 'dirty' samples, even when the input lymphocytes are far below the 1e6 target. The staining doesn't manifest as significant over-titering, and it's not possible to save money by reducing input reagents, because of the need to saturate non-specific binding sites.
  5. We routinely let fresh blood samples sit variable times (1-6 h) at room temperature, before processing, without noticeable effect on our CFC results. We have been forced by occasional circumstance to use blood left overnight (or shipped overnight), and have noticed a variable qualitative and quantitative loss in signal quality.
  6. We have found (unpublished) that PBMC samples that undergo cryopreservation then freezing results in a enrichment of memory phenotypes, and a relative loss of naïve cells. This skewing is variable from sample to sample, and so not easily corrected. It is a significant reason we do nearly all our CFC using freshly-harvested lymphocyte preparations. Other practitioners may not have this option, but need to be aware of this skewing.
  7. Tube-based acquisition from cytometers like the BD LSR-II is specifically designed to use polystyrene tubes (PS). This is because sample is forced up the sip tube by sealing the tube against a gasket, then pressuring the inside of the tube. Polystyrene plastic is rigid, and so pressurizes without deforming. Polystyrene is problematic, however, for any process that cultures immune cells in contact with it. Monocytes (MO) react to polystyrene by trying to phagocytose it, effectively flattening and adhering to the plastic. This complicates full recovery from any PS tubes used to culture suspensions with MOs. Since MO can be involved in antigen presentation in this assay, the tube plastic was switched to polypropylene, to which MO cannot adhere. This results in a technical complication, in that PP tubes do not have exactly the same dimension as PS tubes, and therefore do not form an airtight seal against the BD LSR-II sip assembly gasket. Using PS tubes with these cytometer therefore requires either transferring the finalized sample from PP to PS tubes, or else refitting the cytometer with a modified-dimension gasket (see Note 45).
  8. As a QA feature, when dealing with dozens, hundreds, or even thousands of tubes, we use tube racks equipped with a 'gripper' feature, which can hold each tube up in its slot. When we begin, the tubes are all pulled up. As we add reagent to a tube, we push it down. This way, we keep track of whether a given tube has been treated in a given step.
  9. Negative controls: If your antigens are all in DMSO, you might choose to put an equivalent amount of DMSO into your Negative control tube(s). This lab often has a diversity of test preparations, and so typically does not add solvent to its Negative control.
  10. Staphylococcus Enterotoxin B is a 'superantigen', capable of inducing a non-specific activation a large (e.g. 25%) fraction of T cells, accompanied by massive cytokine release from these cells. This is an effective positive control for this assay, because a failure to elicit massive TNFa or IFNg release strongly implies a technical mistake in execution. Because of its inherent danger, SEB is categorized as a Select Agent, and requires special paperwork to obtain (and then in only small quantities, ≤5mg). It also must be handled carefully. (Since SEB is possibly difficult to obtain, and requires special handling, a user can opt for any other antigen that researcher might know which is likely to reliably give a positive memory-recall response from the researchers cells. SEB is merely convenient and reliable, if available.)
  11. Because this assay does not have a reliable or consistent population of antigen-presenting cells, it is necessary to fully engage the lymphocyte receptors that would otherwise be triggered by the APCs (Antigen Presenting Cells, like macrophages, et al.). In the early work developing this assay, Picker, et al. evaluated the utility of a slate of antibodies against these costimulatory targets. They found that no single antibody target reliably yielded maximum effect, but that the combination of both anti-CD28 and anti-CD49d did.
  12. If a user of this assay wants to incorporate CD28 into the staining panel, a conjugated form of the antibody can be used, with one major caveat: Commercial off-the-shelf conjugated antibody reagents contain an amount of azide preservative that can be toxic to the cells in this assay. To get around this, we contracted for a custom high concentration (2 mg/ml) conjugate in low (0.05%) azide. (We obtained our custom stock from Beckman Coulter. More recently, we are revisiting the issue of whether the amount of azide in off-the-shelf reagents affects ICS results. Preliminary results suggest that amount of azide may not matter, but this testing is incomplete. Please email this author for future updates about this issue.)
  13. There is some controversy about the necessity of staining for the activation marker CD69. Some prominent groups have opted to omit this stain, and instead use the channel for another cytokine, or other marker. In our experience, however, this can result in data artifacts due to cytokine production from unactivated cells. Given the choice to add another cytokine, or ensure the cytokine data is reliable, we've opted for data reliability.
  14. Anti-CD45 is a very useful antibody to include in cases of 'dirty' samples. Example1: In lung wash, lymphocytes are a minority (1-6%) population, and mucous-trapped debris and non-WBC epithelial cells predominate. Example 2: In thawed cryopreserved PBMC, a large fraction of acquired events can be subcellular debris released from fractured cells. Example 3: In enzymatically-digested tissues like gut or liver, lymphocytes may be a minority population, and the process may have generated a lot of debris. In all these cases, an early gate of CD3 vs CD45 quickly removes the debris that would otherwise obscur downstream gating. In our experience, CD45 is required when doing plate-based ICS, because in the shorter fluid column of a plate well, all material (cells and debris) centrifuge to the bottom in a 2-3 min spin. In 5 ml tubes, in contrast, a 10 min spin does not reliably move all the small debris into the pellet. Over the multiple spins of this assay, tube-based ICS becomes largely clarified of small debris, whereas plate-based ICS never does.
  15. Staining for CD107ab offers a way to monitor the degranulation of CTL cells (cytotoxic lymphocyte). Use of this antibody necessitates swapping monensin for Brefeldin A. In our experience, this swap results in a relative reduction in cytokine signals.
  16. DMSO solutions melt slowly at room temperature, and so this reagent needs to be taken out to thaw at least 15 min before use. Moreover, DMSO is somewhat viscous, and so measurements of 1 μl are hard to do accurately. For this reason, and because of the advantages of using a repeater for delivery, we usually prepare dilutions of BfA in our tissue culture medium (49 μl R10: 1 μl BfA), and deliver 50 μl/tube. This volume will form a heavy enough drop on the side of a tube to reliably travel to the bottom of the tube. It is therefore possible to use a repeater, even touching the top of a tube, without great risk of cross-contamination. Such use of a repeater makes it possible to scale up the tube-based method.
  17. FACS Lysing Solution is a reagent invented by BD to do three things simultaneously: (1) Decontaminate a sample (e.g. HIV), (2) rupture and remove RBC (whose multi-sized aggregates can obscure and confound scatterplots, and (3) kill and fix the WBC of interest. FACS Lysing Solution does this by combining a hypotonic (weak) saline with paraformaldehyde (the small, fast-penetrating monomer of formaldehyde). The BD innovation (that continues making them money) was to titer these two components together, such that RBC swell and burst before they fix, whereas WBC fix before they burst. It is important to be be reliably consistent in the timing of this reagent application, because all unpermeabilized, intact cells in a hypotonic solution will continue to swell, thus the scatterplot size will change, if the Lyse is left on longer than 10 min. Perm does not have this problem, because porous cells will not swell due to osmotic pressure.
  18. Many investigators use the one-step reagent, BD CytoFix/CytoPerm, instead of the two-step Lyse, then Perm, so several comments about this are warranted. (1) CytoFix/CytoPerm is an appropriate reagent when dealing with samples lacking a significant presence of RBC (e.g. lung lavage, 'clean' PBMC, lymphocyte harvests from lymph node or gut biopsy). In our work the monkeys are often very sick due to complications of SIV infection, and hemolysis can result in very 'bloody' PBMC harvests. BAL samples can also sometimes be bloody. Given that our research almost always includes blood, our processing necessarily always requires RBC clearance, so we must use 'Lyse'. We therefore consistently apply it to all suspensions, regardless of the strict need. (2) Nonetheless, when we have compared the CFC results obtained from RBC-lacking samples processed with Lyse, then Perm, versus CytoFix/CytoPerm, we found that the former consistently gave larger signals than the latter (though the difference was small, typically a margin of approximately 10%). We attribute this better outcome to the irreversible Tween-20 being a superior permeant than the reversible saponon. (3) When we developed our plate-based CFC method, we realized that the well volumes were insufficient to allow RBC lysis within the wells. We therefore used ACD to remove RBCs prior to adding the cells to the plate. To counter that additional step, and to simplify the process, we substituted CytoFix/CytoPerm for the now-unnecessary 'Lyse'.
  19. The Picker Lab has worked for years with Tritech Research, a maker of non-water-jacketed CO2 incubators, in the development of a 37 °C, CO2 incubator that can rapidly cool to refrigerator temperature at specified clock times, in a way controlled and recorded by a computer. This was prompted by the frequent need, imposed by this CFC protocol, to transfer samples from a conventional incubator to a refrigerator, in the middle of the night (e.g. 3 am). Tritech is now selling a third generation of a single-chamber unit sized for tube racks (called the "UniBator"), and the third generation of a unit with six independently-controllable chambers for plate-based CFC (called the "HexaBator"). Although many incubators are available that can change temperature over a time frame of hours, as far as I know these are the only units capable of dropping from 37 °C to 4 °C in approximately 20 min (as required for consistent CFC results). As of this writing, Tritech has supplied units to the Picker Lab [3 UniBators, 3 HexaBators (all generations)], IAVI (1 UniBator, Gen1), VGTI-Florida/Watkins Lab (1 UniBator, Gen3), and VGTI-Oregon/Sacha Lab (1 UniBator, Gen3).
  20. Many researchers routinely use thawed cells for the CFC assay. Many use 'rest' periods after thawing, but before assay setup, to allow cells triggered for apoptosis to complete this process. Since we do nearly all our CFC with fresh cells, a reliable description of a post-thaw 'rest' and viability assessement can be found at Reference 2.
  21. As a general practice, all the reagents we use in the setup of this assay are equilibrated to room temperature before exposed to cells. A key parameter in our use of this assay is activation, and significant temperature changes to the cells (post-thaw) can affect their degree of activation. Also, the quality of this assay can be degraded by the use of tissue culture medium degraded in L-glutamine, or buffer-exhausted. So as a general practice, we use fresh-made medium for our setups.
  22. Adjustable pipettors are most-accurate in the middle of their range. When measuring 50 μl, for example, a P100 is a better choice than a P200 or P50. Also, DMSO solutions (e.g. peptide preparations) are more viscous than acqueous solutions, and so samples should be drawn and dispensed more slowly than acqueous solutions. Lastly, DMSO solutions have a tendency to form droplets on the outside of pipet tips, so after drawing in a measured amount of reagent, the tip should be dragged up the side of the vessel, to leave behind outside-adhering droplets. Failure to measure antigen accurately can result in large variations in outcomes.
  23. Angling the rack so that the tubes are nearly horizontal is crucial. At this orientation, the depth of the culture is shallower, facilitating better gas exchange. Also, the cells settle out over a much broader surface that would be the case if the tubes were upright. This appears to be a reason why tubes demonstrate better low-end sensitivity than plates (though this has not been experimentally proven). One hypothesis is that the looser, more-dispersed pellet that forms along the side of a tube is more optimal for CFC than is a more-compact pellet, as might form at the bottom of a U- or V-bottomed plate well.
  24. The 1 h pre-BfA interval is only necessary for non-peptide antigens, like proteins or lysates. This interval allows time for some processing to occur before adding the cytoskeleton-disrupting BfA. In unpublished examination, disrupting the pellet by vortexing after adding the BfA had a small, but minor negative effect on the size of antigen responses.
  25. In extensive but unpublished work, the Picker Lab examined the kinetics of antigen-stimulated cytokine secretion. We found that for TNFa, IFNg, and MIP1b, a sigmoidal accumulation of BfA-trapped cytokine signal plateaued from approximately 8-12 h. Beyond 12 h, signal decreased and non-Ag background noise increased. On the basis of this work, we've chosen to always run our CFC assays for 9 h total (1 h prior to BfA, 8 additional h with BfA). This timing yielded the most-consistent large signals for TNFa, IFNg, and MIP1b. In those same kinetic experiments, we found that IL2 signal emerges more quickly, maximizing from 4-6 h after setup, and degrading quickly after 6 h. For this reason, when we do CFC for which the IL2 signal is a very important parameter, we opt to do a 6 h total incubation, instead of a 9 h total incubation.
  26. This write-up describes four distinct ways of removing supernatants from tubes, because this is one of the dominant hand-manipulations involved in this workflow, and different methods are appropriate for different scale-ups. How one removes the supernatant, and how well, is vitally important to the quality of the produced data. The factors deserving attention include: (1) How much residual fluid is left behind? (2) Does the method disrupt and lose any of the cell pellet? (3) Does the method 'clean' the sample of sub-cellular debris? (4) Is the method fast? (5) Does the method require a lot of repetitive hand manipulation?
  27. We use a two-flask vacuum aspirator setup similar to that depicted below:


    Figure 4. Aspirator trap assembly for use in a biosafety cabinet

    In this setup, the aspirator wand goes to a primary trap, which has an overflow tube going to a secondary trap. The vacuum line connecting the secondary trap to the vacuum source has an in-line filter that stops air passage if it gets wet. This way, the vacuum pump never sucks sample material. The important reality to appreciate is that an aspiration wand causes significant air turbulence near its tip, and so to avoid mixing fluids near the pellet, the aspiration process must occur smoothly and fast. The operator should not 'dwell' near the pellet, or else the pellet will mix into the supernatant, and eventually be sucked up. One way to minimize this mixing is to affix a micropipettor tube (without aerosol plug) to the end of an aspiration wand. This significantly reduces air turbulence near the pellet, but it also significantly slows aspiration speed. A fast, effective hybrid approach is to combine use of a 'stopped' wand (see next comment), with a secondary 'clean-up' with a wand affixed with a pipettor tip.


    Figure 5. Aspiration wand, with pipettor tip for finer suction control

  28. An 'stopped' aspirating wand is easily constructed by finding some Tygon tubing with an internal diameter close to the outer diameter of an aspirating wand. A short segment of this tubing can be split, so as to easily move it along the length of the wand. Trial and error can quckly identify the stop at which a smoothly-used wand leaves just the right amount of residual fluid, without any turbulence-stirring of the cell pellet. Once that stop location is determined, the split Tygon tubing stop can be taped shut, and prevented from moving with additional tape (see figure below). A stopped wand removes supernatant very quickly, and the tubes can be left in the centrifuge bucket insert, or rack, during this process. It still does one tube at a time, however, which may still be too slow and labor-intensive when dealing with hundreds of tubes simultaneously, however. Which is an argument for decanting (see next comment).


    Figure 6. Stop on an aspirator wand, to enable rapid, 'no-look' aspiration when handling dozens or hundreds of tubes (note that this figure is better viewed at a 90o angle).

  29. Decanting is just as it sounds, but is an art in practice. The keys to directly pouring out supernatant lie in (1) ensuring the centrifuge spin was long enough to set a tight, affixed pellet, (2) the subsequent processing doesn't 'bump' or vibrate the waiting tubes, loosening them from the tube bottom, (3) the pouring is smooth and fast and consistent in time and motion (leaving little time for the topmost material in the pellet to flow down and drain out of the tube), and (4) just the right amount of 'tap' to the drained tube to disengage the drop left at the end of the tube. Once mastered, the great benefit of decanting is that multiple tubes can be decanted simultaneously, and much faster than other methods. The potential pitfalls are sample loss and splashing (a big concern when dealing with biohazardous materials).


    Figure 7. Hand-decanting supernatant from centrifuged ICS tubes

  30. It is possible to scale this up even more radically by immobilizing all the tubes into a bucket insert using craft foam, as depicted in the figure below. Craft foam (available from arts-and-craft stores) can be easily cut to fit a bucket insert. Our inserts come as stackable layers, and we put foam between two of these layers. Where a tube would go, we first cut an 'X' bigger than the tube, then use a leather punch to remove a hole slightly smaller than a tube. This means that you can easily insert a tube, but that once inserted, the foam holds the tube in the slot. This way, an entire bucket of tubes can be dumped simultaneously. Afterwards, the entire bucket can be vortexed as a unit, but the process is even more an art than decanting handfuls of tubes. Crucially, it is important to know that the tubes at the edges of the insert vortex more effectively than those in the middle, so the insert needs to be vortexed in multiple,varying orientation for the process to be effective in all pellets.


    Figure 8. One example of how ICS tubes can be immobilized in a centrifuge bucket insert, so that decanting and vortexing can be consistently applied to an entire bucket of tubes simultaneously

  31. Resuspending the pellets effectively, especially just prior to adding fixative (Lyse, Perm), is crucial to data quality. As with supernatant removal, we use several different methods, each appropriate to a particular scale of tube handling. For relatively few tubes, we hold several tubes in one hand, and flick/drag them over the fingers of the opposite hand several times (e.g. three times in one orientation, then rotate 90 degrees and draw them over again another three times). This is a very effective 'gold standard'. Alternatively, it is possible to drag the bottoms of the tubes against the front grate of a biosafety cabinet, rotating the tubes after half the drags, and this can achieve the same effect. An operator can also vortex individual tubes against a vortexer cup, or handfuls of tubes against a vortexer plate, but it is important to remember that if the tubes are held upright, the vortex created has its 'eye-of-the-hurricane' situated over the pellet one wants to disrupt. Therefore, one should always vortex the tubes at a (changing) angle, ideally after an initial flick to dislodge the pellet from the floor of the tube. Scaling up further, one can vortex an entire rack, or a gasketed bucket insert (like that depicted above), but this involves 'art', and one should practice it with pelleted RBCs to ensure one knows just how much vortexing, and how much orientation-changing is needed to ensure the pellets are fully and reliably disrupted.
  32. We long ago confirmed that cocktails of antibodies work as well as individually-administered single doses of antibody. Cocktails have the advantages of less hand-work, and more accurate measurements (large volumes are better-measured than small volumes). Moreover because staining kinetics is dependent on antibody concentration, we realized that consistent-volume cocktails give consistent results. For this reason, we typically formulate our cocktails such that we dispense 50 μl per tube. 50 μl reliably runs down the side of a PP tube, and is a volume effectively dispensed by a repeater.
  33. Initially, we always did staining on ice, in the dark. As we scaled up, this was one of the 'nice-ities'that was abandoned. When I investigated whether this has any negative effects on our quantitative results, I found none. Moreover, the staining is mostly done within 20 min, so a 30 min incubation is not sacred. Thirty min produces reliably-consistent good results, works well with large-scale processing, and allows for reasonable breaks (if needed).
  34. The Lyse step is the one that most-determines staining quality. If the cell pellet is ineffectively-disrupted, the paraformaldehyde will chemically fix clumped cells &/or cells and debris together. It also crucially affects the morphology of the lymphocytes, and hence the appearance of the key scatterplot display. The perm step tends not to be so pivotal, because fixed cells seem not to clump as avidly as live cells.
  35. This is a potential stopping point, if needed. At this point, the cells are fixed, and the paraformaldehyde is diluted, and so the tubes can sit days (e.g. a weekend) at 4 °C without any significant loss in quality. If we develop cytometer problems that will delay acquisition, we stop our stains at this point, so that at least the intracellular stains are as 'fresh' as possible before acquisition.
  36. It is not essential to disrupt the cell pellets afterwards. The goal is primarily to dilute the residual paraformaldehyde and Tween-20, and the volume held in the interstitial space is negligible to this process.
  37. It is important to do two washes, because the overall goal is to reduce-to-innocuous the amount of protein-fixing paraformaldehyde before adding the intracellular stain antibodies (which are proteins).
  38. Ki67 is a protein that transiently appears in the nucleus during mitosis. The staining pattern is a smear (rather than distinct negative and positive populations), and so obtaining useful timepoint comparisons is hugely dependent on the staining being absolutely consistent from timepoint to timepoint. To get this stain optimal, and consistent, it is necessary to use the smallest, brightest fluorophore (FITC, which penetrates to the nucleus best), and enough staining time (45 min) so that the staining process is reliably complete. Lastly, it is important that the samples be washed afterwards, with enough time in wash fluid so that anti-Ki67 MAb that has not bound to high-affinity sites has enough time to drift out of the cell (by the way, this is a disadvantage of CytoFix/CytoPerm, which requires the continuous presence of the permeabilizer,saponin, for intracellular staining. Once saponin is removed, whatever is inside the membrane is trapped. When Tween-20 is used, the membranes are dissolved away, such that low-affinity binding is lost during sample storage time, prior to acquisition. So sample quality can improve slightly over a day of storage).
  39. In the past,we added 1% paraformaldehyde to the tubes, to fix the intracellular antibodies in place. We came to realize this was not necessary (the high-affinity clones we were using had no noticeable off-rate in the < 3 days storage time), and that the addition of any fluid just increased our cytometer acquisition times.
  40. The exact purpose of beta-Mercaptoethanol is a bit unclear, and I've seen several different explanations. One is that it is a reducing agent that fights free radical oxygen species affecting lymphocytes. Another is that bME enhances the cysteine supply to the lymphoid cells, thereby increasing the intracellular level of tripeptide glutathione (Meister and Anderson, 1983). All seem to agree, however that empirically, bME improves tissue culture studies of lymphocytes (at least murine lymphocytes).
  41. CD45RA identifies naïve T cells, and is a stain recommended by the NHP ICS standards group, in their report of a plate-based method (Donaldson et al., 2012).
  42. CCR7 is found on T cells that require a secondary stimulus prior to displaying effector function. As with CD45RA, this is a stain recommended by the NHP ICS standards group (Donaldson et al., 2012).
  43. The Invitrogen Aqua LIVE/DEAD reagent stains protein, and distinguishes live and dead cells on the basis of how much protein the dye can access. Cells have approximately 1/100 as much protein on their surface as inside. A live cell has an intact membrane, and therefore takes up far less stain than a porous dead cell, which typically stains 2 logs brighter than a live cell.
  44. Cell phenotype markers like CD3, CD4, and CD8 can be stained either during the surface stain, or during the intracellar stain (CD45 MAb does not work on PFA-fixed antigen, so must be applied as a surface stain). For CD3 and CD8, there are both benefits and consequences staining these markers at the intracellular step. On the positive side, both receptors undergo internalization during antigen engagement, so responding cells become 'dimmer' in a surface stain. An inexperienced analyst could gate these cells out, unless s/he took into account the reduced surface brightness of responding cells. Applying these stains intracellularly obviates this problem, since the internalized receptors remain, and are available for staining. The drawback to this approach, however, is that all intracellular stains reduced the intensity separation between negative and positive populations.


    Figure 9. How intracellular staining of CD3+ T cells can solve the problem of CD3 internalization upon activation

    Positive populations tend to stay put, but the negative population tends to be brighter for intracellular stains, because more low-affinity binding sites are available to antibodies accessing the insides of cells.

  45. Most flow analysers push sample suspension up into their sip tube by pressurizing the sample tube. Because polystyrene is rigid, many analysers were designed for polystyrene tubes. On such cytometers (e.g. BD LSR-II), the tubes are fitted onto a sip assembly, with the tube wall forming an airtight seal against a gasket.


    Figure 10. Illustration of the sip assembly of a BD LSR-II cytometric analyser, showing the gasket at the top, which is designed to seal against polystyrene tubes

    Unfortunately, since monocytes and macrophages adhere themselves to PS, it is not appropriate for the CFC assay, since after a culture period any adherent cells will be unavailable for analysis. Consequently, a non-'sticky' plastic like polypropylene (PP) should be used (see Note 7). Standard PP tubes have a slightly different diameter than PS tubes, however, so the standard gasket on the sip assembly does not form an airtight seal against PP tubes. One easy solution to this problem is to widen a standard gasket by adding the outer ring of a second gasket, as illustrated below:


    Figure 11. How an LSR-II sip assembly gasket can be modified to seal against polypropylene tubes (the type of plastic best for ICS)

  46. In our experience, conventional CFC samples are fairly robust at 4 °C storage. Since the MAb-identified populations are mostly separated and discrete, the 'defocussing' that occurs during storage takes a while to affect gating outcomes. Our rule is that these samples should be acquired within 72 h, though use of some less-stable fluorophores might shorten this time. The inclusion of 'smeared' populations (e.g. Ki67, CD95) can also make it necessary to acquire sample data sooner.
  47. Because the possibility always exists that the cytometer will be unavailable (out-of-service, or fully-occupied) when samples need to be collected, we have investigated whether full-stained samples can be frozen, then thawed after a cytometer becomes available again. We have been surprised that this can sometimes work, though the scatterplot changes slightly. In our experience, most fluorescent results show no qualitative or quantitative changes. It is crucial to test this out before relying on this desperation move, however, because we have found that some fluorescent stains are, in fact, significantly changed. If one wanted this as a reliable fallback, various panels should be tested beforehand, and only ones robust through a freeze-thaw cycle should be used.

QA/QC Notes

'QA' stands for Quality Assurance, and 'QC' stands for Quality Control. QA refers to how you set a process up so as to maximize consistent quality, and minimize errors (or the consequence of mistakes). QC refers to post-process checking of the process outcome, to identify quality errors, and alert you that QA needs to be improved.
Some QA/QC notes relevant to the CFC assay:

  1. Training
    1. Prepare SOPs (Standard Operating Procedures).
    2. Have competent people train those who are learning.
    3. Proficiency test those who have been trained.
      1. Compare the outcomes of those who are competent with those learning.
      2. Compare the outcomes, occasionally, of those deemed competent (to identify method 'drift' issues that affect quality or consistency).
      3. Change the SOP to reflect current best practices.
  2. Reagents
    1.  All antibody reagents should be re-titered against the kind of cells you'll be staining (e.g. blood, PBMC, BAL, etc.; using an N ≥ 4).
    2. All process buffers should be at room temperature, and NOT warmed from 4 °C with a waterbath set at 37 °C. The reason for using buffers all at room temperature is (1) to avoid temperature shocks that induce activation, and (2) to keep the mammalian cells below the temperature at which they're metabolically active. Room temperature reliably does both.
  3. Cells
    1. Freshly-harvested cells give more consistent results
      1. Thawed cells are variable for many reasons
      2. Variable effectiveness at cryopreservation
      3. Variable storage time, quality
      4. DMSO toxicity
      5. Variable thawing quality
        Keep cells from warming to metabolic activity when in the presence of DMSO
      6. Broken cell debris generated by the freeze-thaw (F/T) cycle, and adhering to live cells
      7. Intact carcasses of F/T-killed cells seeming like live cells (and artificially increasing the 'non-responding' denominator population
      8. Naïve cells are preferentially (but variably) lost during the F/T cycle
    2. The CFC assay is designed for 1e6 Ly/tube, but works as well for 0.5-2e6 Ly/tube, in 1 ml culture. If you reduce the culture volume, medium exhaustion and waste buildup in active cultures can variably affect result quality intra-run.
  4. Sample ID, labeling
    1. Always arrange samples in a logical, reproducible, consistently-applied order
      1. e.g. Our monkey IDs are 5-digit numbers
        A random order: 25113, 25131, 23115, 25311
        Numerically-ordered: 23115, 25113, 25131, 25311
        This way, you can always ensure they're in a logical, reproducible order
    2. Give samples two names, one that's a simplified shorthand easier for users to use
      1. e.g. A = 23115, B = 25113, C = 25131, D = 25311
      2. Or, if they're all in 'Group A': A1 = 23115, A2 = 25113, A3 = 25131, A4 = 25331
      3. This way, operators can deal with (A,B,C,D) or (A1,A2,A3,A4) instead of those confusing 5-digit numbers (which are just begging to get mixed-up).
      4. Also, if one number in an array is mis-transcribed, it is possible to deduce the error and correct it.
    3. Number-identify antigens in a similar way
      1. e.g. 1 = Neg, 2 = X, 3 = Y, …7 = SEB
      2. In this way, if you orient the tubes in rack by this order, the first tube you handle will always be the negative control, and the last one will always be the antigen most likely to cause cross-contamination problems.
      3. This dual-identification,as noted above, helps definitively correct transcription errors.
      4. And this dual-identification has benefits in FlowJo and Excel.
        FlowJo recognizes only the first 32 characters of a filename. If the 32nd character is 2=, you have a unique identification of that sample in a way you wouldn't if you were instead listing several SIV_( ) antigens.
        After you export data from FlowJo, you end up with a filename which is a long text string, in which multiple discrete pieces of information are embedded. The Excel tool 'Text-To-Data' is useful for 'exploding' filenames into component parts, and characters like '=' are handy for this process.
    4. Use computer-printed labels
      1. Use a serif font (so that lower-case 'L' isn't confused with '1', for example).
      2. Use a font size large enough to be easily read.
      3. Use understood abbreviations, to allow for larger font sizes.
      4. Use label color to communicate grouping, timepoint, monkey, antigen, etc.
  5. Process
    1. Use tube-gripping racks
      'Tube UP' means step (e.g. cocktail addition) has not yet happened.
      'Tube DOWN' means step completed.
    2. Move reagent tubes laterally, or back/forward in their rack, to indicate whether they've been used yet.
    3. Prepare a checklist of steps, or reagents,and mark them off as the reagents are used, or steps completed.
    4. Use label placement on tubes to consistently orient them in racks.
      1. Reagent 1 can be dispensed down one wall.
      2. Reagent 2, during a following step, can be repeater-dispensed down a different wall, with reduced likelihood of cross-contamination.
    5. Organize samples so that the negative control tubes are always processed first, and the most-potent-antigen tubes are always processed last. This minimizes the consequences of any cross-contamination that does happen.
    6. Always process tubes in the same order. That increases the likelihood that they'll all get similar duration for each process (e.g. Lyse treatment, antibody exposure, etc).
    7. Make reagent cocktails, rather than delivering individual doses. Larger measurements are more accurate, and the fewer the measurements you make, the less likely you'll make a mistake.
    8. Habitually drag micropipettor tips up the side of a tube when you pull it out of a reagent vial. That way, you drag off any fluid holding on to the outside of the pipettor tip. This is especially important when measuring very small volumes of peptide antigen.
    9. Pipettors are most-accurate in their midrange. Use a P100 to measure 50 μl, rather than a P200 or P50.
    10. If possible, set up multiple negative controls, and use an average value for any background-subtraction.
    11. If confronted with so many samples that the processing cycle is too long to maintain quality, break the work up into parts that can be done with high quality. Sometimes, this issue applies to only a single step. For example, the CFC step most likely to cause quality problems is the addition of Lyse. If necessary, do sub-batches at a time, so as to maintain quality.
  6. Stain
    1. Use validated staining panels.
    2. Put the brightest fluorophores on the rarest events.
    3. Put the least-effective fluorophores on the most-plentiful &/or most-intense events.
    4. Be aware of issues like the spectral bleed of PE-Cy7 into the PE channel, and so put PE-Cy7 on rarer, dimmer events.
    5. Staining volume matters, so the use of consistently-50 μl cocktail volumes makes results more consistent.
  7. Acquisition
    1. Samples should be acquired within 72 h.
    2. In our experience samples stained with Alexa-700 suffer rapid quality erosion, and should be acquired within 24 h.
    3. If cytometer backlog or dysfunction mean some CFC samples will wait longer than 72 h before acquisition, the staining process can be stopped after Lyse,and the intracellular stain delayed until just before acquisition.
  8. Equipment
    1. Each piece of critical equipment should be assigned to a staff member, who is given responsibility to monitor, maintain, performance-assess, and repair.
    2. Coulter counters reliably count only blood samples, and are notoriously inaccurate for anything else. They can make hugely-variable, hugely-inaccurate counts with debris-rich samples like BAL, thawed cells, and enzymatically-digested solid tissues.
    3. Flow cytometers:
      1. Cytometers should be monitored by the BD CST system (or a functionally equivalent performance-monitoring system).
      2. Lasers should be warmed at least 45 minutes before use.
      3. Fluidics should get cleaned at least once a day (and again, if a lot of 'dirty' samples are run, leading to buildups that change the time delay).
      4. Compensation:
        Use compensation beads, rather than stained cells (Beads are brighter.).
        Make fresh compensation bead samples weekly.
        Make compensation samples for all antibody reagents used (This is especially important for the tandem dyes, like PerCP-Cy5.5, PE-TexasRed, PE-Cy7, APC-Cy7, etc.).
        Compensate each experiment anew (rather than using compensation matrices from weeks before).
      5. Acquisition speed matters
        Discrete populations can gateably-tolerate 'blurring' better than smears. Therefore CFC without CD28 v CD95, or Ki67 gating can be run faster with that gating.

Recipes

  1. Aqua LIVE/DEAD kit
    1. Concentrated dye stock
      1 vial powder dissolved in 50 μl DMSO, can stored as frozen aliquots for weeks
    2. Staining Solution (made fresh)
      1. Dilute 2 μl conc dye into 78 μl deionized water (DI)
      2. Dilute 50 μl of the solution in step 5bi into 950 μl 1x PBS
      3. Stored on ice prior to use
  2. 'R10' tissue culture medium
    1. RPMI-1640 (1x) (w/o L-glutamine, 0.1 μm filtered)
    2. Fetal Bovine Serum (defined, heat-inactivated, 40 nm-filtered)
      Stored as frozen aliquots
    3. Penicillin+Streptomycin (P/S) Solution
      Stored as frozen aliquots
    4. L-glutamine (200 mM)
      Stored as frozen aliquots; light-sensitive when thawed
    5. Sodium pyruvate (SP)  
    6. beta-Mercaptoethanol (bME) (Note 40)
      Light sensitive; stored in foil-wrapped bottle
    7. Sterile-filtration apparatus 500 ml capacity 0.22 μm cellulose-acetate filter
    8. Formulation
      1. Thaw frozen components
      2. Combine additives in top of filter system, then filter
        50 ml FBS
        10 ml P/S
        10 ml L-glutamine
        5 ml SP
      3. Rinse filter with sterile RPMI
      4. Directly pour additional RPMI into the filtrate, to 500 ml
      5. While swirling the R10, slowly add 500 μl bME
      6. Cap tightly; protect from light; stored at 4 °C
  3. 'PAB' (phosphate albumin buffer) wash buffer
    1. DPBS, makes 10 L at 1x, or 1 L at 10x
    2. Bovine serum albumin (BSA)
    3. Sodium azide (preservative; NaN3)
    4. Highly toxic (Wear mask when measuring!)
    5. Deionized, filtered water (because of the azide, autoclaving not needed)
    6. Formulation of 10x stock:
      1. Dispense 4 L of DI water into a 10 L carbuoy
      2. Add a sterilized stirbar
      3. A top a magnetic stirrer, start a gentle swirl
      4. Add 50 g of BSA slowly to on the top of the swirl (avoid clumps)
      5. Add 22.75 g of NaN3, slowly
      6. Stir overnight, at room temperature
      7. Next day, after all previous additives are dissolved
      8. Add contents of five bottles of powdered DPBS (each bottle makes 1 L at 10x)
      9. Rinse bottles with fluid from the carbuoy, then return
      10. Increase stir speed; check every 2 h until all is dissolved
      11. Dispense 10x solution into sterile 1 L bottles; cap tightly
      12. Store bottles at 4 °C
    7. Diluting to 1x working stock
      1. Into an emptied and sterilized 20 L carbuoy, pour two 1 L bottles of 10x PAB stock
      2. Rinse the emptied 1 L bottles with DI water, return
      3. Add DI water to carbuoy until the total volume is 20 L
      4. Cap, then mix by inversion
      5. Stored carbuoy at 4 °C
      6. Using 1x working stock
      7. Transfer to bottle with pump dispenser (e.g. Brinkmann Bottletop Dispenser; e.g., Brand Tech Dispensette)    
      8. Use PAB at room 4 °C or room temperature
  4. 'Lyse' fixation and RBC-lysing solution (Note 17)
    1. BD FACS Lysing Solution, 10x concentrate
      Note: Contains paraformaldehyde.
    2. Deionized water
    3. Formulation
      1. Combine ingredients in the following proportions
      2. 50 ml 10x FACS Lysing Solution
      3. 450 ml DI water
      4. Mix by shaking, stirring, or inversion
      5. Dispense into dark plastic bottles
      6. Store tightly capped bottles at room temperature
      7. QC test with whole blood for acceptable outcome
  5. 'Perm' (1x) fixation and irreversible cell-permeating solution (Note 18)
    1. 1x 'Lyse' (from above)
    2. Tween-20 (polyoxyethylenesorbitan monolaurate)
    3. This requires a high-viscosity pipettor to measure well
    4. Formulation
      1. To 1 L of 'Lyse' add 500 μl Tween-20 while stirring
      2. Stir atop a magnetic stirrer overnight
      3. Dispense into dark plastic bottles
      4. Store tighly capped bottles at room temperature
      5. QC test with Ki67 antibody, for acceptable outcome

Acknowledgments

The methods described in this protocol have been evolving since the 1995 paper cited in this protocol (Picker et al., 1995), and were used in the two 2013 papers published by our group (Hansen et al., 2013a and Hansen et al., 2013b). Funding has been provided by NIH and by the Bill and Melinda Gates Foundation.

References

  1. Andersson, U., Hallden, G., Persson, U., Hed, J., Moller, G. and DeLey, M. (1988). Enumeration of IFN-γ-producing cells by flow cytometry. Comparison with fluorescence microscopy. J Immunol Methods 112(1): 139-142.
  2. Donaldson, M. M., Kao, S. F., Eslamizar, L., Gee, C., Koopman, G., Lifton, M., Schmitz, J. E., Sylwester, A. W., Wilson, A., Hawkins, N., Self, S. G., Roederer, M. and Foulds, K. E. (2012). Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies. J Immunol Methods 386(1-2): 10-21.
  3. Foulds, K. E., Donaldson, M. and Roederer, M. (2012). OMIP-005: Quality and phenotype of antigen-responsive rhesus macaque T cells. Cytometry A 81(5): 360-361.
  4. Fukazawa, Y., Park, H., Cameron, M. J., Lefebvre, F., Lum, R., Coombes, N., Mahyari, E., Hagen, S. I., Bae, J. Y., Reyes, M. D., 3rd, Swanson, T., Legasse, A. W., Sylwester, A., Hansen, S. G., Smith, A. T., Stafova, P., Shoemaker, R., Li, Y., Oswald, K., Axthelm, M. K., McDermott, A., Ferrari, G., Montefiori, D. C., Edlefsen, P. T., Piatak, M., Jr., Lifson, J. D., Sekaly, R. P. and Picker, L. J. (2012). Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines. Nat Med 18(11): 1673-1681.
  5. Gardner, M. B. (1989). SIV infected rhesus macaques: an AIDS model for immunoprevention and immunotherapy. Adv Exp Med Biol 251: 279-293. 
  6. Hansen, S. G., Vieville, C., Whizin, N., Coyne-Johnson, L., Siess, D. C., Drummond, D. D., Legasse, A. W., Axthelm, M. K., Oswald, K., Trubey, C. M., Piatak, M., Jr., Lifson, J. D., Nelson, J. A., Jarvis, M. A. and Picker, L. J. (2009). Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat Med 15(3): 293-299.
  7. Hansen, S. G., Ford, J. C., Lewis, M. S., Ventura, A. B., Hughes, C. M., Coyne-Johnson, L., Whizin, N., Oswald, K., Shoemaker, R., Swanson, T., Legasse, A. W., Chiuchiolo, M. J., Parks, C. L., Axthelm, M. K., Nelson, J. A., Jarvis, M. A., Piatak, M., Jr., Lifson, J. D. and Picker, L. J. (2011). Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine. Nature 473(7348): 523-527.
  8. Hansen, S. G., Sacha, J. B., Hughes, C. M., Ford, J. C., Burwitz, B. J., Scholz, I., Gilbride, R. M., Lewis, M. S., Gilliam, A. N., Ventura, A. B., Malouli, D., Xu, G., Richards, R., Whizin, N., Reed, J. S., Hammond, K. B., Fischer, M., Turner, J. M., Legasse, A. W., Axthelm, M. K., Edlefsen, P. T., Nelson, J. A., Lifson, J. D., Fruh, K. and Picker, L. J. (2013). Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science 340(6135): 1237874.
  9. Hansen, S. G., Piatak, M., Jr., Ventura, A. B., Hughes, C. M., Gilbride, R. M., Ford, J. C., Oswald, K., Shoemaker, R., Li, Y., Lewis, M. S., Gilliam, A. N., Xu, G., Whizin, N., Burwitz, B. J., Planer, S. L., Turner, J. M., Legasse, A. W., Axthelm, M. K., Nelson, J. A., Fruh, K., Sacha, J. B., Estes, J. D., Keele, B. F., Edlefsen, P. T., Lifson, J. D. and Picker, L. J. (2013). Immune clearance of highly pathogenic SIV infection. Nature 502(7469): 100-104.
  10. Jung, T., Schauer, U., Heusser, C., Neumann, C. and Rieger, C. (1993). Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159(1-2): 197-207.
  11. McClure, H. M., Anderson, D. C., Fultz, P. N., Ansari, A. A., Lockwood, E. and Brodie, A. (1989). Spectrum of disease in macaque monkeys chronically infected with SIV/SMM. Vet Immunol Immunopathol 21(1): 13-24.
  12. Picker, L. J., Singh, M. K., Zdraveski, Z., Treer, J. R., Waldrop, S. L., Bergstresser, P. R. and Maino, V. C. (1995). Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86(4): 1408-1419.
  13. Pitcher, C. J., Hagen, S. I., Walker, J. M., Lum, R., Mitchell, B. L., Maino, V. C., Axthelm, M. K. and Picker, L. J. (2002). Development and homeostasis of T cell memory in rhesus macaque. J Immunol 168(1): 29-43.
  14. Prussin, C. and Metcalfe, D. D. (1995). Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods 188(1): 117-128.
  15. Sander, B., Andersson, J. and Andersson, U. (1991). Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119: 65-93.
  16. Schuerwegh, A. J., Stevens, W. J., Bridts, C. H. and De Clerck, L. S. (2001). Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1β, interleukin-6, and tumor necrosis factor-alpha in monocytes. Cytometry 46(3): 172-176.
  17. Waldrop, S. L., Pitcher, C. J., Peterson, D. M., Maino, V. C. and Picker, L. J. (1997). Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J Clin Invest 99(7): 1739-1750.

简介

最初称为"CFC"(细胞因子流式细胞术)的现在更通常称为"ICS"(细胞内染色),或更不常称为"ICCS"(细胞内细胞因子染色)。关键的创新是使用有效的渗透物(允许细胞内染色)和试剂破坏分泌(捕获细胞因子,从而能够累积可检测的细胞内信号)。因为不是所有使用该技术的研究人员都对细胞因子感兴趣,'ICS'术语已经受到青睐,尽管这里将使用"CFC"。
CFC是细胞功能的测试,将淋巴细胞暴露于培养物中的抗原,然后测量任何诱导的细胞因子应答。处理测试培养物以用用荧光标记物标记的单克隆抗体染色细胞,并使用多聚甲醛化学固定细胞和净化样品。
CFC提供流式细胞术的功能,包括批量采样和多参数互相关,用于抗原特异性记忆反应的分析。使用CFC的研究者能够表型表征用测试抗原和表型亚型(例如,CD4 + 或CD8 + T细胞)培养的细胞,确定高于背景水平的产生频率的细胞因子。
与ELISPOT和Luminex方法相反,CFC可以关联来自特定的,表型 - 表征的细胞的多种细胞因子的产生。 CFC测定可用于检测个体具有抗原暴露(如在群体筛选中),或用于跟踪抗原记忆的出现和持续(如在疫苗接种,感染或发病机理的研究中)。除了量化抗原响应细胞的%频率外,平均荧光强度可用于评估响应细胞中产生多少细胞因子。随着流式细胞术的技术进步,目前的CFC用户常常可以获得11个荧光通道(或甚至18个),使得可以高度表征抗原反应细胞的表型,或者同时定量根据许多细胞因子或活化标记的反应。强大的软件如FlowJo(TreeStar)和SPICE(NIAID)可用于分析数据,并对细胞因子反应进行复杂的多变量分析。
此处描述的方法针对恒河猴的细胞进行定制,注释笔记代表了十年的积累的技术经验。相同的方案容易应用于其它哺乳动物细胞(例如人或小鼠),尽管确切的抗体克隆将根据宿主系统而不同。这里描述的基本方法在染色和固定之前,在布雷菲德菌素A的存在下,用抗原和共刺激抗体孵育1ml管培养物中的1×10 6个淋巴细胞。

关键字:ICS, CFC, 流式细胞仪, ICCS, 抗原

[历史背景] Andersson等人于1989年完成了固定和透化淋巴细胞的第一次报告,然后用针对IFNγ的抗体染色。在1991年,Sander 证明了改进的方法,使用多聚甲醛固定细胞,皂苷(两亲性糖苷)使其透化,荧光标记的抗体染色细胞内细胞因子用于显微镜检查。 1993年,Jung等人扩展了用于流式细胞术的该方法,包括莫能菌素(阻断细胞内蛋白质转运的聚醚抗生素离子载体)以抑制分泌,从而增加细胞因子的细胞内信号否则将在合成后不久释放的分子。 1995年,Prussin和Metcalfe使用直接偶联的抗体,并且在6小时温育中报道了良好的结果。同样在1995年,Picker等人通过使用布雷菲德菌素A('BfA',一种真菌内酯抗生素)阻断细胞因子分泌装置,以及通过使用不同的透膜物(吐温20),显着增强了细胞因子检测的灵敏度和再现性。这种改进的方法由Picker等人在1997年的报告中应用于人类HIV +患者中的抗原特异性内稳态机制。在2001年,Schuerwegh等人证实BfA在测定中比莫能菌素提供更好的细胞因子信号,尽管莫能菌素在该方法中仍被其他人广泛使用。
关于非人类灵长类动物研究,1989年的两份报告,一份是加德纳,另一份是McClure,表明恒河猴是研究艾滋病毒和艾滋病的有用模式。在2002年,Picker等人报告了对恒河猴进行特殊修饰的CFC测定的应用。 2012年,一个旨在为在罗猴疫苗研究中使用CFC的协作小组建立标准的联合组织指定小组发表了他们对于具有6小时总孵育的96孔板方法的建议(Donaldson等人 2012; Foulds等人,2012)。
这里报道的一般程序是2002管形式(见注1)方法,现在与9小时总孵化,并优化特别是低端敏感性。这里的具体细节是现在在由俄勒冈国家灵长类研究中心附属的俄勒冈健康和科学大学的Picker实验室实施的现有技术。这些方法已经用于我们最近的几个出版物中(Hansen等人,2013a; Hansen等人,2013b,Fukazawa等人)。 >,2012; Hansen等人,2011; Hansen等人,2009)。重要的是要注意,在我们手中,板形CFC对于弱响应不是那么敏感和可再现,如在此描述的基于管的方法(未公开的观察)。直到该差异被理解和解决,基于管的方法仍然是CFC最敏感的格式。

材料和试剂

  1. 来自恒河猴血液(注2)或支气管肺泡灌洗(BAL)的淋巴细胞悬液或从固体活检或尸检组织收获,通过对样品准确的方法测定的细胞密度(注3和4)
    1. 每次测试需要〜1×10 6个存活淋巴细胞
    2. 新鲜获得(注5)
    3. 或解冻的冷冻保存的样品(注6)
  2. 抗原
    1. 阴性对照(注释9)
    2. 积极控制:
      超敏抗原葡萄球菌肠毒素B SEB(注10)(Toxin Technology,目录号:BT202)(冻干粉,100μg;储备液:100μg/ml,在水中;用量:2μl/试验)
    3. 其他阳性对照(实验特定)
    4. 肽混合物(1-100种不同的肽,≥2μg/肽/1ml-试验) 15个氨基酸肽(15mer),由11个氨基酸重叠
  3. 抗体
    1. 培养孵化期间共刺激的非共轭抗体(注11)
      抗CD28,纯的非结合的,克隆CD29.2(注12)
      抗CD49d,纯非共轭,克隆9F10
      稀释至0.5mg/ml的储液; 使用1μl每1 x 10 6 Ly
    2. 基本荧光团结合的单克隆抗体
      的   您使用的荧光团取决于流式细胞仪可用   you。 许多公司出售合适的荧光团 抗体,包括BD,Beckman Coulter,Life Technologies, Invitrogen TM ,eBiosciences等 抗CD3e,与恒河猴反应的克隆(SP34-2,FN18)
      抗CD4(L200,MT477))
      抗CD8a(SK1,RPA-T8)
      抗CD69(FN50,CH/4,TP1.55.3)(注13)
      抗IFNg(B27)
      抗TNFα(MAB11)
    3. 可选荧光团共轭单克隆抗体
      抗CD45(DO58-1283)(注14)
      抗IL2(MQ1-17H12)
      抗MIP1b(D21-1351)
      蚂蚁-CD107(α:H4A3,β:H4B4)(注15)
      抗CD95(DX2)
      抗CD45RA(L48,5H9,MEM-56,其他)(注41)
      抗CCR7(CD197)(注42)
      抗Ki67(B56)(注38)
  4. 布雷菲德菌素A(Sigma-Aldrich,目录号:B-7651) 供应商: (Sigma-Aldrich,目录号:B-7651; BioLegend,目录号:91850)
    工作原料:10mg/ml,在DMSO(1.0μl/试验)(注16)
  5. Benzonase(Merck KgaA,Novagen)(使用50U/ml)
  6. 1×RPMI-1640(w/o L-谷氨酰胺,0.1μm过滤)(例如HyCone,目录号:SH30096.02)
  7. 胎牛血清(FBS,aka:'FCS')(例如HyClone,目录号:SH30070.03)(定义为,热灭活,40nm过滤)
  8. 青霉素+链霉素(P/S)溶液(例如Sigma-Aldrich,目录号:P-0781)
  9. L-谷氨酰胺(200mM)(例如Sigma-Aldrich,目录号:G-7513(100ml))中。
  10. 丙酮酸钠(SP)(例如Sigma-Aldrich,目录号:S-8636)(100ml)
  11. (巯基乙醇(bME)(例如Sigma-Aldrich,目录号:M-7522)(100ml)(注40)
  12. 无菌过滤装置(例如Corning,目录号:430769)(500ml容量0.22μm纤维素乙酸酯过滤器)
  13. Dulbecco's磷酸盐缓冲盐水(DPBS)(例如Thermo Fisher Scientific,Corning,目录号:55-031-PB)
  14. 牛血清白蛋白(BSA)(例如Thermo Fisher Scientific,目录号:BP1605100)
  15. 叠氮化钠(防腐剂; NaN 3)(例如Thermo Fisher Scientific,目录号:BP922-500)
  16. BD FACS溶解液(10倍浓缩物)(BD,目录号:349202)
  17. 吐温-20(聚氧乙烯山梨聚糖单月桂酸酯)(例如Sigma-Aldrich,目录号:P-7949)
  18. Aqua LIVE/DEAD试剂盒(Life Technologies,Invitrogen TM www.lifetechnologies.com ,搜索"LIVE/DEAD"用于一系列不断变化的污渍和试剂盒(注43)
    1. 浓缩染料(见配方)
    2. 染色溶液(新鲜)(见配方)
  19. 组织培养基('R10')(参见配方)
  20. 'PAB'洗涤缓冲液(见配方)
  21. "溶解"固定和RBC溶解溶液(参见配方)(注17)
  22. 'Perm'固定和细胞透化溶液(见配方)(注18)

设备

  1. 管(注1和7)聚丙烯(PP)(圆底,5ml/12×75mm,无菌)(例如 Falcon ,目录号: 2054)
  2. 计算机生成的管道打印标签(可选;必须很好地粘到聚丙烯)
  3. 管托架(可选)(例如 Thermo Fisher Scientific,No-Wire夹具架10-13 MM 90位置)(注8)
  4. 泡沫化妆品楔子(或功能等同物)(我们使用的是躺在长边上的2"长x 3/4"高。)
  5. 层流生物安全柜(用于不育,即使不与病原体一起工作)
  6. 收集的真空吸气器
  7. 合适的流体测量分配器,带有适当的一次性用品
    1. 电动泵移液器,用于测量1-50 ml
    2. 用于0.5-1,000μl之间的手动手动微量移液器
    3. 中继器(例如来自Eppendorf的)
    4. 手持转发器,用于0.5-2 ml之间的测量
    5. 固定泵分配器,用于1-5毫升之间的测量
  8. 带有旋转铲斗(能够800 x g )的离心机(例如 Sorvall Legend T/RT)
  9. Vortexer
  10. 实验室计时器
  11. 用于组织培养的培养箱(加湿,在37℃,5%CO 2气氛下稳定)
    选项:标准水夹套T/C培养箱(例如Thermo Fisher Scientific,Forma TM ,Series II,Model:3110)
    选项:UniBator(Tritech Research)DigiTherm CO <2>培养箱,带快速冷却和双向接口(注19)
  12. 在4°C冰箱
  13. 流式细胞术分析仪,6-荧光检测器或更多(例如BD,型号:LSR-II)
  14. 计数PBMC(例如,,Coulter计数器,Guava或血球计数器)的方法

程序

注意:请参见图1和图2,分别为CFC设置过程和CFC管染色过程的流程图。


图1. CFC设置流程
的流程图

图2. CFC管染色过程的流程图

文化设置,孵化,冷却

  1. 预启动准备
    1. 请参阅质量保证/质量控制说明。
    2. 使新鲜介质(或开始升温至室温)。
    3. 标签PP管。
    4. 在DMSO中开始解冻肽(如果适用)。
  2. 获取细胞
    1. 新鲜收获的WBC(注释2-5)。
    2. 来自血液的PBMC(外周血单核细胞)。 (其他方案解释如何从血液中分离PBMC。也可以使用已经用低渗盐水如ACK处理的全血来爆发红细胞。)
    3. BAL(支气管肺泡灌洗,筛分)
    4. 实体组织,机械加工(LN,脾,骨)
    5. 固体组织,酶处理(肠,肝,阴道)
    6. 冷冻保存的WBC收获
      1. 解冻(注20)。
      2. 休息(注20)。
      3. 确定活力(注20)。
  3. 在新鲜制备的R10(注21)中将悬浮液密度调节至约2×10 6 Ly/ml,
    1. 例如。 1 x 10 6 Ly/500μ。
    2. R10 - 由抗体或抗体补充。
    3. 将悬浮液置于室温下备用,直到使用。
  4. 制备补充了协同刺激MAb抗CD28,抗CD49d的R10(注11)
    1. 确定测定装置所需的总体积:(#管)×(500μl/管)×1.2(即,增加20%)。
    2. 将R10测量到标有"costim-R10"的容器中
    3. 加入抗CD28和抗CD49d:
      1. 假定[stock] = 0.5mg/ml
      2. 使用1μl/500μl(,即,2μl/ml R10)
      3. 混合(涡旋或反转)。
  5. 准备R10 + costim的子库,补充抗原
    1. 确定特定抗原(#管)×(500μl/管)×1.1(即,增加10%)所需的总体积。
    2. 在由抗原标记的血管中测量R10 + costim
      1. 简单涡旋原料抗原是混合和均匀的
      2. 确保测量准确(注22)。
      3. 混合(涡旋或反转)。
  6. 在所有适当标记的管中分配500μlR10 + costim + Ag
    1. 使用管标签可以在保持架中以可再现的方式一致地定向管
    2. 在使用前立即涡旋溶液(使用中继器进行分配是方便的)
    3. 分配500μl/管,瞄准管的特定"侧面"(当使用中继器时,该侧面现在是交叉污染的可能原因,因此后续步骤应该使管子定向不同,以避免交叉污染)。
    4. 将管保持架放入37℃,5%CO 2的培养箱中,直到使用(这些管可以在加入细胞前2小时制备)。
  7. 分配500微升的细胞悬浮液到所有搁架管
    1. 注意管子上的标签方向; 定向管使得分配的悬浮液沿管的相对侧下降(以最小化交叉污染的风险)
    2. 在此步骤中使用中继器非常方便。
    3. 始终先分配到"负极"控制管
    4. 始终分配到"SEB"阳性对照管。
    5. 该步骤应该尽可能快地进行,因为它将细胞与抗原组合。 然而,在室温下,细胞尚不具有代谢活性
    6. 在这种组合之后,不需要单独涡旋管
  8. 将该架子在37℃,5%CO 2下稳定地转移到培养箱中
    1. 确保加湿盘含有水(或者从管中蒸发将浓缩细胞,Ag和盐,改变测定结果)。
    2. 将机架放在侧面,将机架顶部放在化妆品楔上。 管的尖锐角度使得介质更浅,使得气体扩散更容易。 这种取向还使得细胞沉降在更大的区域上,具有更少的细胞 - 细胞接触。 这似乎提高了反应敏感性,原因仍不清楚
    3. 设置计时器1小时。


      图3.如何在孵育期间对一个ICS管架放置

  9. 孵育1小时后,向所有管中加入布雷菲德菌素A(注24)
    1. 准备稀释鸡尾酒,以50微升/管使用。
      1. 如果(BfA原液)为10mg/ml,使用1μl/管
      2. 将BfA稀释为未补充的R10。 1份BfA加入39份R10; 将40μl该混合物输送到每个ICS管中。 40μl液滴将可靠地在PE管的壁上滑落。
    2. 使用中继器,将BfA向下射入管中。
      1. 定位标签以最小化交叉污染。
      2. 分配以使每个管中沉降的细胞团的破裂最小化
      3. 40微升产生足够大的液滴,可靠地从管的侧面流下
    3. 不要涡旋或进一步混合管内容物
    4. 管子应该不加盖(或用松散的"卡帽"),重要的是允许容易的气体交换。在短期培养期间,钢笔/链霉菌通常对于防止细菌和真菌生长是非常有效的。
  10. 将架子返回到培养箱(加湿,37℃,5%CO 2)
    1. 孵育管额外5-8小时(机架倾斜如图3)(注25)。
  11. 在孵育结束时,应当冷却机架
    1. 最简单的方法是将机架转移到4°C的冰箱
    2. Tritech Research销售新型孵化器,通过计算机时钟控制,可以快速冷却(37°C至15°C,不到20分钟;最终达到4°C)(注19)。

第二部分。 染色

  1. 样品应在12小时内加工固定
    1. 尽管哺乳动物细胞在4℃下代谢上几乎是惰性的,但它们仍然存活。 离开太长,CFC信号将改变。
    2. 将培养管从培养架转移到离心机铲刀插入件
    1. 使用瓶口泵分配器,用PAB清洗缓冲液填充所有管
    2. 离心机800×g,10分钟(足以在5ml管中产生紧密的细胞沉淀)。 运行的温度无关紧要,只要它远低于代谢活性37℃; 单元格将很快被固定
    3. 除去上清液(注释26)
      1. 方法1:吸条抽吸(注释27)。
      2. 方法2:吸棒吸出并停止(注28)
      3. 方法3:倾析(注释29)。
      4. 方法4:使用垫片进行倾析(注释30)。
    4. 重悬颗粒(视频1)(注31)。

      视频1.用CFC(ICS)测定法从聚丙烯管中手洗滗洗上清液
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  2. 用Aqua LIVE/DEAD鉴别剂染料染色(可选,特别适用于解冻细胞)
    1. 用冰冷的1×PBS清洗细胞
    2. 吸出以除去上清液(留下最少的残余流体)
    3. 在100μl冰冷的"染色溶液"(100μl/1×10 6次/孔)中重悬细胞(如部分II,步骤B4)。
      1. 在黑暗中,在冰上孵育20分钟。
    4. 清洗(见第II-B部分)停止。
  3. 应用表面抗体混合物(注32)
    1. 鸡尾酒可以提前几小时在PAB。
    2. 以50μl/管配制鸡尾酒例如:CD45,CD4(注释44)。
    3. 免除中继器。
    4. 将鸡尾酒加入试管后涡旋试管。
    5. 在室温下孵育30分钟(在黑暗中)(注释33)。
    6. 如果表面染色剂包括两阶段生物素 - 链霉亲和素染色,则第一混合物应包括生物素化的试剂。 在30分钟温育后,将链霉亲和素试剂加入管中(不需要预洗涤)。 将链霉亲和素置于50μl/管PAB稀释液中是方便的,这允许中继分配。
  4. 清洗(见第II-B部分)
  5. 每管应用1ml'Lyse'(注17和34)
    1. 设置实验室计时器10分钟。
    2. 确保所有颗粒有效地破裂。
    3. 有一个计划,将允许在两分钟内涡旋,当你开始分配Lyse,并确保管以涡流相同的顺序添加Lyse。
    4. 填写适当大小的中继器。
    5. 迅速分配到试管中。
      1. 在添加到第一个试管之前启动计时器。
      2. 将中继器瞄向中点墙。
    6. 开始涡旋管2分钟内收到Lyse。
      1. 如何添加Lyse是整个过程中最重要的细节之一。 如果细胞在加入Lyse之前没有很好地重悬,它们将被固定在一起,降低数据质量。 如果您在合并细胞后不要快速混合细胞,那么您可能会导致RBC的破裂不良,降低数据质量。 在添加Lyse之后,很快并且强烈地涡旋是很重要的
      2. 如果时间允许,再次涡旋管(如果样品含有RBC污染,这是特别有价值的)
    7. 在室温下,在黑暗中孵育十分钟。
  6. 清洗(见第II-B部分)
    添加PAB降低Lyse试剂的低渗性,减缓细胞的渗透肿胀,并稀释化学固定细胞蛋白质的多聚甲醛。 残余液体将与稀释液相匹配(注释35)。
  7. 每管应用0.5 ml'Perm'(注18)
    基本上,重复与"Lyse"相同的方法,但使用0.5 ml /管
  8. 清洗(注36)
  9. 清洗(注37)
  10. 应用细胞内抗体混合物(注32)
    1. 鸡尾酒可以提前几小时在PAB。
    2. 以50μl/管配制鸡尾酒实例:CD3,CD8,CD69,细胞因子(注释44)。
    3. 免除中继器。
    4. 将鸡尾酒加入试管后涡旋试管。
    5. 在室温下孵育30-45分钟(室温,在黑暗中)(注释33) 如果鸡尾酒包括Ki67,则孵育时间应延长至45分钟(注38)。
  11. 清洗(注39)
    1. 尽可能多地移除流体,以确保快速采集时间。

第三部分。 数据采集

  1. 请参阅有关QA/QC的注意事项。
  2. 样品应在72 h内采集(注释46和47)。

笔记

  1. 这里提出的是专门的管格式方法。尽管确实基于板的方法提供了许多优点(多通道处理,更小的试剂体积,简化的过程,机器人采集),但是我们学习了该方法的某些方面导致低端灵敏度测试抗原。而管和板方法产生高于约0.2%细胞因子反应的可比CFC结果,在我们的手中的管方法一致地执行更好与弱响应低于该水平。我们在2007年认识到这一点,并且还无法修改96孔板方法来实现相同的低端结果。
  2. 当使用血液时,必须使用抗凝血剂,否则感兴趣的细胞将在凝块形成中丧失。 Picker实验室优选ACD(酸 - 柠檬酸盐 - 葡萄糖,"柠檬酸钠")作为抗凝剂。重复评估已经证明(未公开的观察),当使用肝素时,CFC结果在质量上不同,并且定量上较少。在我们的工作中,使用PCR定量释放到细胞外液的病毒,因此不能使用抗凝血剂螯合剂EDTA。否则,它已经被证明在CFC和ACD中都有效
  3. Coulter计数器(例如,在设备部分中指出的Beckman Coulter Ac * T diff5)被设计为量化血液中的人细胞,尽管它们可以针对恒河猴细胞进行校准。然而,该技术不是设计用于含有显着的非亚细胞或亚细胞碎片的样品,如在从解冻中回收的冷冻保存的样品中,或从机械地和/或酶处理以收获淋巴细胞的固体组织活检中可充分的。在任何情况下,不是从新鲜血液开始,库尔特计数器将不准确地报告细胞密度,通常通过超量计数。此外,这种计数器不能可靠地区分活细胞和死细胞。在我们对这个问题的仔细(未发表)检查中,我们发现这些计数器超过淋巴结和脾脏的淋巴细胞收获,通常为1-2倍。来自肺清洗或由酶消化的组织(例如肝脏,肠)产生的淋巴细胞收获在基于该技术的计数器中是功能无法计数的,因为可以填充计数门的残留物的数量非常大。在样品进行冻融的情况下,生存力不能可靠地评估。在这些情况下,其他技术必须增加计数(例如可行性染色),或更换Coulter计数器技术。一种广泛使用的替代方案是Guava计数器,使用一遍具有活死染色(例如 ViaCount),以及另一个通过至少散点图和CD3 + 的选通。这可能是昂贵和耗时的。对于该测定,确切地说,如何计数细胞没有关系,但是用户应当知道,大多数计数方法(甚至血细胞计数器)对于临床非血WBC是不准确的。由于所有这些样品将在流式细胞仪上收集,所述流式细胞仪是能够报告关于所测定的T细胞数目的精确数目的仪器,所以所述测定本身提供了对用于输入的计数方法进行QC的方法。 br />
  4. 该方案中描述的CFC方法假设每次测试输入1×10 6个淋巴细胞,并且抗体和试剂输入是成比例的。然而,在实践中,难以正确地预先量化从支气管嗅觉灌洗(BAL)获得的淋巴细胞或从来自肠或肝组织的长时间的酶促收获,因此使用经验确定的近似。我们已经发现(未公开)在"脏"细胞悬浮液输入(例如,BAL,酶消化)中淋巴细胞与淋巴细胞标记抗体的严格比例性分解。例如,如果Coulter计数报告到CFC管中的BAL输入是1e6淋巴细胞,但是实际上输入是1/20的淋巴细胞,我们发现使用1/20的染色抗体产生定性和定量上更差的结果而不仅仅是假定输入实际上是1e6淋巴细胞。我们假设解释是"脏"样品具有丰富的低亲和力非特异性结合位点,其"大量"了抗体试剂。因此,即使当输入淋巴细胞远低于1e6靶时,这里描述的CFC方法仍然可以有效地用于"脏"样品。染色不表现为显着的过度滴定,并且由于需要饱和非特异性结合位点,不可能通过减少输入试剂来节省金钱。
  5. 我们常规地让新鲜血液样品在室温下变化时间(1-6小时),然后加工,对我们的CFC结果没有显着影响。我们偶尔被迫使用血液过夜(或过夜运送),并注意到信号质量的可变质和定量损失。
  6. 我们已经发现(未公开),经历冷冻保存然后冷冻的PBMC样品导致记忆表型的富集和初始细胞的相对损失。这种偏移随样品而变化,因此不容易校正。这是一个重要的原因,我们做几乎所有我们的CFC使用新鲜收获的淋巴细胞制剂。其他从业者可能没有这个选项,但需要知道这种偏差
  7. 像BD LSR-II这样的流式细胞仪的基于管的采集是专门设计为使用聚苯乙烯管(PS)。这是因为通过将管密封在垫圈上,然后对管的内部加压,将样品强制上升到sip管。聚苯乙烯塑料是刚性的,因此加压不变形。然而,聚苯乙烯对于培养免疫细胞与其接触的任何过程都是有问题的。单核细胞(MO)通过试图吞噬它,与聚苯乙烯反应,有效地平整和粘附到塑料。这使得从用于培养具有MO的悬浮液的任何PS管的完全恢复复杂化。由于在该测定中MO可以参与抗原呈递,所以将管塑料转换成聚丙烯,其中MO不能粘附。这导致了技术复杂性,因为PP管不具有与PS管完全相同的尺寸,并且因此不形成抵靠BD LSR-II套管组件衬垫的气密密封。因此,使用这些细胞仪的PS管需要将最终样品从PP转移到PS管,或者用改变尺寸的垫片重新安装细胞计数器(见注释45)。
  8. 作为一个QA功能,当处理几十个,几百个甚至几千个管时,我们使用配有"夹具"功能的管架,可以将每个管保持在其插槽中。当我们开始,管都被拉起来。当我们向试管中加入试剂时,我们推下它。这样,我们跟踪给定管是否在给定步骤中被处理。
  9. 阴性对照:如果您的抗原都在DMSO中,您可以选择将等量的DMSO放入阴性对照管中。该实验室通常具有多种测试制剂,因此通常不向其阴性对照添加溶剂。
  10. 葡萄球菌肠毒素B是一种"超级抗原",能够诱导大量(例如<25%)T细胞的非特异性活化,伴随着大量细胞因子从这些细胞。这是该测定的有效阳性对照,因为未能引起大量TNFα或IFNg释放强烈意味着执行中的技术错误。由于其固有的危险,SEB被归类为选择代理,并且需要特殊的文书工作以获得(然后仅少量,≤5mg)。它也必须小心处理。 (由于SEB可能难以获得,并且需要特殊的处理,用户可以选择研究者可能知道哪些可能可靠地给研究者细胞产生积极的记忆 - 回忆反应的任何其它抗原,SEB只是方便和可靠,如果可用。)
  11. 因为该测定法不具有可靠或一致的抗原呈递细胞群体,所以有必要充分地接合否则将被APC所触发的淋巴细胞受体(抗原呈递细胞,如巨噬细胞, em>)。在开发该测定的早期工作中,Picker等人评价了针对这些共刺激靶标的抗体片段的效用。他们发现没有单个抗体靶可靠地产生最大效果,但是抗CD28和抗CD49d的组合确实产生。
  12. 如果该测定的用户想要将CD28掺入染色板,则可以使用抗体的缀合形式,一个主要警告:商业现货的缀合的抗体试剂包含一定量的叠氮化物防腐剂,其可以对在该测定中的细胞。为了解决这个问题,我们在低(0.05%)叠氮化物中收缩定制的高浓度(2mg/ml)缀合物。 (我们从Beckman Coulter获得了我们的定制库存,最近我们正在重新研究现有试剂中叠氮化物的量是否影响ICS结果的问题。初步结果表明叠氮化物的量可能无关紧要,但这种测试请发送电子邮件给作者,以获取有关此问题的更新信息。)
  13. 对于激活标记CD69染色的必要性存在一些争议。一些突出的团体已经选择省略这种染色,而是使用通道另一种细胞因子或其他标记。然而,在我们的经验中,这可能导致由于未活化细胞产生细胞因子而导致的数据假象。鉴于选择添加另一种细胞因子,或确保细胞因子数据可靠,我们选择了数据可靠性
  14. 抗CD45是包括在"脏"样品的情况下非常有用的抗体。实施例1:在肺洗涤中,淋巴细胞是少数(1-6%)群体,并且粘液捕获的碎片和非WBC上皮细胞占优势。实施例2:在解冻的冷冻保存的PBMC中,大部分获得的事件可以是从裂解细胞释放的亚细胞碎片。实施例3:在酶消化的组织如肠或肝中,淋巴细胞可以是少数 人口,这个过程可能产生了大量的碎片。在所有这些情况下,CD3对CD45的早期门快速移除否则将遮挡下游门控的碎片。在我们的经验中,当进行基于板的ICS时,需要CD45,因为在板孔的较短流体柱中,所有材料(细胞和碎片)在2-3分钟旋转中离心至底部。相比之下,在5ml管中,10分钟的旋转不能将所有小碎片可靠地移动到沉淀中。在该测定的多次旋转中,基于管的ICS变得在很大程度上澄清了小碎片,而基于板的ICS从未变得明显。
  15. CD107ab的染色提供了监测CTL细胞(细胞毒性淋巴细胞)脱颗粒的方法。使用该抗体需要将莫能菌素换成布雷菲德菌素A.在我们的经验中,这种交换导致细胞因子信号的相对减少。
  16. DMSO溶液在室温下缓慢熔化,因此该试剂在使用前需要至少15分钟解冻。此外,DMSO有些粘稠,因此1μl的测量很难准确。由于这个原因,并且由于使用中继器的交付的优势,我们通常准备稀释的BfA在我们的组织培养基(49微升R10:1微升BfA),并提供50微升/管。该体积将在管的侧面上形成足够可靠的液滴,以可靠地行进到管的底部。因此,可以使用中继器,甚至接触管的顶部,而没有交叉污染的巨大风险。这种中继器的使用使得可以放大基于管的方法
  17. FACS溶解溶液是由BD发明的一种试剂,其同时进行三项操作:(1)净化样品(例如HIV),(2)破裂并除去RBC(其多种尺寸的聚集物可以遮蔽和混淆散点图和(3)杀死和固定感兴趣的WBC FACS裂解溶液通过组合低渗(弱)盐水和多聚甲醛(甲醛的小,快速穿透单体)来实现这一点。BD创新(继续使他们赚钱)是将这两种组分滴定在一起,使得RBC在它们固定之前肿胀和爆发,而WBC在它们爆发之前固定。重要的是在该试剂应用的时间上可靠地一致,因为所有未穿透的,完整的细胞低渗溶液将继续膨胀,因此,如果Lyse留在超过10分钟,散点图大小将改变。Perm不会有这个问题,因为多孔细胞不会 由于渗透压而膨胀
  18. 许多研究者使用一步法试剂,BD CytoFix/CytoPerm,而不是两步Lyse,然后Perm,因此有关这一点的几个意见是有保证的。 (1)当处理缺乏RBC显着存在(例如肺灌洗,"清洁"PBMC,来自淋巴结或肠活检的淋巴细胞收获物)的样品时,CytoFix/CytoPerm是合适的试剂。在我们的工作中,猴子由于SIV感染的并发症而常常非常恶心,溶血可导致非常"血腥"的PBMC收获。 BAL样品有时也可能是血色的。鉴于我们的研究几乎总是包括血液,我们的处理必然总是需要RBC清除,所以我们必须使用"Lyse"。因此,我们一直将其应用于所有悬浮液,无论严格的需要。 (2)尽管如此,当我们比较了从RBC缺乏的样品(用Lyse,然后Perm,CytoFix/CytoPerm)获得的CFC结果时,我们发现前者一致地给出比后者更大的信号(尽管差异很小大约10%的余量)。我们将这个更好的结果归因于不可逆的吐温-20是比可逆皂苷更优越的渗透性。 (3)当我们开发基于板的CFC方法时,我们意识到孔体积不足以允许RBC在孔内裂解。因此,我们在将细胞加入板中之前使用ACD去除RBC。为了应对这个额外的步骤,并简化过程,我们用CytoFix/CytoPerm代替现在不必要的"Lyse"。
  19. Picker实验室已经与Tritech研究公司合作多年,该公司是一家无水夹套的CO 2 sub孵化器的制造商,在37℃,CO 2 sub孵化器的开发中,可以以指定的时间时间快速冷却至冰箱温度,以由计算机控制和记录的方式。这是由于通过该CFC方案强加的频繁需要,在半夜(例如上午3点)将样品从常规培养箱转移到冰箱。 Tritech现在销售第三代尺寸适合管架的单室单元(称为"UniBator"),第三代单元具有六个独立可控室用于基于板的CFC(称为"HexaBator") 。虽然许多孵化器都可以改变温度在一段时间的时间框架内,据我所知,这是唯一能够在大约20分钟内从37°C下降到4°C(根据一致的CFC结果所需的)。截至本文,Tritech已向Picker Lab [3 UniBators,3 HexaBators(all generation)],IAVI(1 UniBator,Gen1),VGTI-Florida/Watkins Lab(1 UniBator,Gen3)和VGTI-Oregon/Sacha Lab(1 UniBator,Gen3)。
  20. 许多研究人员通常使用解冻的细胞用于CFC测定。许多在解冻后使用"休息"期,但在测定设置之前,允许触发细胞凋亡的细胞完成该过程。由于我们用新鲜细胞几乎完成了所有CFC,所以可以在参考文献2中找到关于解冻后休息和活力评估的可靠描述。
  21. 作为一般实践,我们在该测定的设置中使用的所有试剂在暴露于细胞之前平衡至室温。我们使用这种测定法的一个关键参数是激活,并且细胞的显着的温度变化(解冻后)可以影响它们的激活程度。此外,该测定的质量可以通过使用在L-谷氨酰胺中降解的组织培养基或缓冲液耗尽来降解。因此,作为一般做法,我们使用新鲜的介质为我们的设置
  22. 可调式移液器在其范围的中间是最精确的。例如,当测量50μl时,P100是比P200或P50更好的选择。另外,DMSO溶液(例如肽制剂)比水溶液更粘稠,因此应当比水溶液更慢地抽取和分配样品。最后,DMSO溶液具有在移液管尖端的外部形成液滴的倾向,因此在吸入测量量的试剂后,尖端应当沿着容器的侧面拖动以留下外部粘附的液滴。未能准确测量抗原可能导致结果的巨大变化
  23. 使支架成角度使得管几乎水平是关键的。在该取向,培养物的深度更浅,有利于更好的气体交换。此外,细胞沉积在更宽的表面上,如果管是直立的情况。这似乎是为什么管表现出比板更好的低端敏感性的原因(虽然这没有经实验证明)。一个假设是,沿着管的侧面形成的较松散的,更分散的丸粒比在U形或V形底部板孔的底部形成的更紧密的丸更适合于CFC。 br />
  24. 1 h pre-BfA间期仅对非肽抗原,如蛋白质或裂解物是必需的。该间隔允许在添加细胞骨架破坏BfA之前进行一些处理的时间。在未公开的检查中,在加入BfA后通过涡旋破碎沉淀对抗原反应的大小具有小的,但是较小的负面影响。
  25. 在广泛但未发表的工作中,Picker实验室检查了抗原刺激的细胞因子分泌的动力学。我们发现对于TNFa,IFNg和MIP1b,BfA捕获的细胞因子信号的S形积累从约8-12小时平台。超过12小时,信号减少和非Ag背景噪声增加。在这项工作的基础上,我们选择总是运行我们的CFC测定共9小时(在BfA之前1小时,用BfA另外8小时)。这个时间产生了对TNFa,IFNg和MIP1b最一致的大信号。在这些相同的动力学实验中,我们发现IL2信号更快地出现,从设置后4-6小时最大化,并且在6小时后快速降解。因此,当我们做IL2信号是非常重要的参数的CFC时,我们选择6小时总孵育,而不是9小时总孵育。
  26. 这个写作描述了从管中去除上清液的四种不同的方式,因为这是在该工作流中涉及的主要手操纵之一,并且不同的方法适合于不同的放大。如何去除上清液,以及多好,对生产数据的质量至关重要。需要注意的因素包括:(1)剩余流体剩余多少? (2)方法是否破坏并丢失任何细胞沉淀? (3)方法是否"清洁"亚细胞碎片样品? (4)方法快吗? (5)该方法是否需要大量的重复手动操作?
  27. 我们使用类似于下面描述的两烧瓶真空吸气器设置:


    图4.用于生物安全柜的吸气器捕集阱

    在这个设置中,吸气棒流向主捕集阱,其具有通向第二捕集阱的溢流管。将次级捕集器连接到真空源的真空管线具有在线过滤器,如果其变湿则停止空气通道。这样,真空泵从不吸取样品材料。要理解的重要现实是,抽吸棒在其尖端附近引起显着的空气湍流,并且因此避免在丸粒附近混合流体,抽吸过程必须平稳和快速地进行。操作者不应该在沉淀附近"停留",否则沉淀会混入上清液中,最终被吸出。使这种混合最小化的一种方法是将微量移液器管(无气雾剂塞)固定到抽吸棒的端部。这显着减少了球团附近的空气湍流,但也显着减慢了吸气速度。快速,有效的混合方法是结合使用"停止"的鞭子(见下一个评论),与辅助'清理'与附加吸管器尖端的魔杖。


    图5.吸液管,带有用于更好吸力控制的移液器吸头

  28. 通过找到一些内径接近吸气管外径的聚乙烯管,可以很容易地构造"停止"吸气管。该管的短段可以分开,以便容易地沿着棒的长度移动它。试验和错误可以快速识别停止,其中平滑使用的棒仅留下适量的残余流体,而不对细胞沉淀物进行任何湍流搅拌。一旦确定了停止位置,分流聚乙烯管道停止装置可以关闭胶带,并使用额外的胶带防止移动(参见下图)。停止的棒非常快速地除去上清液,并且在此过程中管可以留在离心机桶插入件或架中。然而,它仍然一次只做一个管,然而,当同时处理数百个管时,这仍然可能太慢和劳动密集。这是一个滗析的参数(见下一个注释)。


    图6.停止在吸气棒上,以便在处理数十或数百根管子时能够快速,"无视"吸气(注意,这个数字在90° o 角度时更好) 。

  29. 滗析就像它的声音,但在实践中是一种艺术。直接倒出上清液的关键在于(1)确保离心机旋转足够长以设置紧密的,固定的丸粒,(2)随后的处理不会使等待的管子"碰撞"或振动,从管子中松开底部,(3)倾倒是平滑和快速的并且在时间和运动上一致(使得颗粒中的最顶层材料几乎没有时间向下流动并从管中排出),以及(4)恰好适量的" '到排出的管以使在管的端部处留下的液滴脱离。一旦掌握,倾析的巨大好处是多个管可以同时倾析,并且比其他方法快得多。潜在的缺陷是样品损失和飞溅(当处理生物危害材料时是一个很大的问题)

    图7.离心的ICS管的手动倾析上清液

  30. 通过将所有管固定到使用工艺泡沫的桶插入件中,可以进一步扩大规模,如下图所示。工艺泡沫(可从工艺品商店获得)可以容易地切割成适合桶插入物。我们的插件是可堆叠的层,我们把泡沫放在这些层之间。在管子将要去的地方,我们首先切割一个比管子大的"X",然后使用皮革冲头去除比管子略小的孔。这意味着您可以轻松插入管,但一旦插入,泡沫将管保持在插槽中。这样,整个桶的管可以同时倾倒。之后,整个桶可以作为一个单元进行涡流,但是该过程甚至是一种艺术,而不是倾倒少量的管。重要的是,重要的是要知道插入件边缘处的管比那些在中间的管更有效,因此插入件需要在 多重,变化的方向,使过程对所有颗粒都有效

    图8. ICS管如何固定在离心机铲斗插入件中的一个示例,以便滗析和涡旋可以同时连续施加到整桶管子上

  31. 有效地重新悬浮颗粒,特别是在添加固定剂(Lyse,Perm)之前,对数据质量至关重要。与上清液去除一样,我们使用几种不同的方法,每种方法适合于特定规模的管处理。对于相对少的管,我们在一只手中握持几个管,并且在相反的手的手指上轻拂/拖动它们几次(例如在一个方向上三次,然后旋转90度并将它们拉过再次三次)。这是一个非常有效的"黄金标准"。或者,可以将管的底部拖曳到生物安全柜的前格栅上,在拖动一半之后旋转管,这可以实现相同的效果。操作者还可以使单个管相对于涡流器杯涡旋,或者少量管抵靠涡流器板涡旋,但是重要的是要记住,如果管保持直立,则产生的涡流具有其"飓风之眼"位置在球员一个想破坏。因此,人们应该总是以(改变)角度涡流管,理想地在初始轻拂之后将颗粒从管的底部移出。进一步扩大,可以涡旋整个机架,或一个垫圈的斗式插入(如上所述),但这涉及"艺术",并且应该用颗粒RBC实践它,以确保一个知道多少涡流,多少需要改变方向以确保颗粒完全可靠地破碎
  32. 我们早就证实,抗体的鸡尾酒与单独施用的单剂量抗体一样好。鸡尾酒具有手工工作量少,测量精度更高的优点(大体积比小体积测量更好)。此外,因为染色动力学取决于抗体浓度,我们意识到一致的体积鸡尾酒给出一致的结果。因此,我们通常配制我们的鸡尾酒,使我们分配每管50微升。 50μl可靠地从PP管的一侧流下,是一个由中继器有效分配的体积
  33. 最初,我们总是在冰上,在黑暗中染色。当我们扩大规模,这是一个"好,it''at被遗弃。当我研究这是否对我们的定量结果有任何负面影响,我没有发现。此外,染色大多在20分钟内完成,因此30分钟的孵育不是神圣的。三十分钟产生可靠一致的良好结果,适用于大规模加工,并允许合理休息(如果需要)。
  34. 溶解步骤是最确定染色质量的步骤。如果细胞沉淀物被无效地破坏,多聚甲醛将化学地将结块的细胞和/或细胞和碎片固定在一起。它还关键影响淋巴细胞的形态,因此关键散点图显示的外观。 perm步骤往往不那么重要,因为固定的细胞似乎不像活细胞一样积聚。
  35. 如果需要,这是一个潜在的停止点。在这一点上,细胞被固定,并且多聚甲醛被稀释,因此管可以在4℃下放置几天(例如例如周末),而没有任何显着的质量损失。如果我们开发会延迟采集的细胞计数器问题,我们在这一点停止我们的污渍,以至于在获得之前至少细胞内染色是"新鲜的"。
  36. 之后破坏细胞沉淀物不是必需的。目标主要是稀释残留的多聚甲醛和吐温-20,并且保留在间隙空间中的体积对于该过程可以忽略不计。
  37. 重要的是进行两次洗涤,因为总体目标是在加入细胞内染色抗体(其是蛋白质)之前减少至无害的蛋白质固定的多聚甲醛的量。
  38. Ki67是在有丝分裂期间短暂出现在细胞核中的蛋白质。染色模式是涂片(而不是不同的阴性和阳性群体),因此获得有用的时间点比较极大地依赖于从时间点到时间点绝对一致的染色。为了使染色最佳和一致,必须使用最小,最亮的荧光团(FITC,其最好地渗透到细胞核)和足够的染色时间(45分钟),使得染色过程可靠地完成。最后,重要的是,随后洗涤样品,在洗涤液中有足够的时间,使得没有结合到高亲和力位点的抗Ki67 MAb具有足够的时间漂移出细胞(顺便说一下,这是一种CytoFix/CytoPerm的缺点,它需要持续存在透化剂皂甙,用于细胞内染色。一旦去除皂苷,则膜内部被截留,当使用Tween-20时,膜被溶解掉,在样品储存期间,在采集前,残余结合会丢失,因此样品质量可能在储存一天后略有改善)。
  39. 在过去,我们添加1%多聚甲醛到管,以固定细胞内抗体到位。我们意识到这不是必要的(我们使用的高亲和力克隆在< 3天储存时间内没有明显的解离速率),并且任何流体的添加只是增加了我们的细胞计数器获取时间。 >
  40. β-巯基乙醇的确切目的有点不清楚,我看到了几个不同的解释。一个是它是一种与影响淋巴细胞的自由基氧物质竞争的还原剂。另一个是,bME增强半胱氨酸向淋巴样细胞的供应,从而增加三肽谷胱甘肽的细胞内水平(Meister和Anderson,1983)。所有人似乎都同意,但是经验上,bME改善淋巴细胞(至少是鼠淋巴细胞)的组织培养研究。
  41. CD45RA鉴定了幼稚的T细胞,并且是由NHP ICS标准组在其基于板的方法的报告中推荐的染色剂(Donaldson等人,2012)。
  42. 在显示效应子功能之前需要次级刺激的T细胞上发现CCR7。与CD45RA一样,这是NHP ICS标准组推荐的污渍(Donaldson等人,2012年)。
  43. Invitrogen Aqua LIVE/DEAD试剂染色蛋白,并根据染料可以获得多少蛋白质区分活细胞和死细胞。细胞在其表面上具有与内部相比大约1/100多的蛋白质。活细胞具有完整的膜,因此比多孔死细胞污染少得多,其通常比活细胞污染2个日志更亮。
  44. 细胞表型标记如CD3,CD4和CD8可在表面染色期间或在细胞内染色(CD45MAb不作用于PFA固定的抗原,因此必须作为表面染色)上染色。对于CD3和CD8,在细胞内步骤染色这些标记物有益处和后果。在积极方面,两种受体在抗原接合期间经历内化,因此反应细胞在表面染色中变得"调暗"。没有经验的分析人员可以选择这些细胞,除非他/他考虑到响应细胞的表面亮度降低。在细胞内应用这些污渍消除了这个问题,因为内化的受体保留并且可用于染色。然而,这种方法的缺点是所有细胞内染色降低了阴性和阳性群体之间的强度分离

    图9.如何CD3 + T细胞的细胞内染色可以解决激活后CD3内化的问题

    阳性人群倾向于保留,但阴性人群对细胞内染色趋于更亮,因为更多的低亲和力结合位点可用于接近细胞内部的抗体。

  45. 大多数流量分析仪通过对样品管加压将样品悬浮液向上推入其sip管。由于聚苯乙烯是刚性的,许多分析仪被设计用于聚苯乙烯管。在这样的细胞仪(例如BD LSR-II)上,管被装配到套管组件上,其中管壁对垫圈形成气密密封。


    图10. BD LSR-II细胞计数分析仪的sip组件图示,显示顶部的垫圈,用于密封聚苯乙烯管

    不幸的是,由于单核细胞和巨噬细胞自身粘附于PS,因此不适合用于CFC测定,因为在培养后,任何贴壁细胞将不可用于分析。因此,应使用非粘性塑料如聚丙烯(PP)(见注7)。标准PP管的直径稍微不同于PS管,但是,sip组件上的标准垫圈不能形成对PP管的气密密封。这个问题的一个简单的解决方案是通过添加第二个垫圈的外圈来加宽标准垫圈,如下所示:


    图11.如何修改LSR-II吸嘴组件垫圈以密封聚丙烯管(最适合ICS的塑料类型)

  46. 根据我们的经验,常规CFC样品在4℃储存时相当稳健。由于MAb鉴定的群体大多是分离的和离散的,在存储期间发生的"散焦"需要一段时间来影响 门控结果。我们的规则是这些样品应在72 h内采集,但使用一些不太稳定的荧光团可能会缩短这一时间。包含"涂抹"群体(例如,Ki67,CD95)也可能使得有必要更快地采集样品数据。
  47. 因为当需要收集样品时,细胞计数器总是存在不可用(停止服务或完全占用)的可能性,所以我们调查了是否可以冷冻全染色的样品,然后在细胞计数器再次可用后解冻。我们惊讶的是,这有时可以工作,虽然散点图略有变化。根据我们的经验,大多数荧光结果显示没有定性或定量的变化。然而,在依赖于这种绝望移动之前测试这是至关重要的,因为我们已经发现,一些荧光染料事实上显着改变。如果想要这是一个可靠的后备,应该事先测试各种面板,并且只有通过冻融循环稳健的面板应该使用。

QA/QC注释

'QA'代表质量保证,'QC'代表质量控制。 QA是指如何设置一个过程,以便最大限度地保持质量,并尽量减少错误(或错误的后果)。 QC是指对过程结果进行后处理检查,以识别质量错误,并提醒您质量检查需要改进。
与CFC测定相关的一些QA/QC注释:

  1. 训练
    1. 准备SOP(标准操作程序)。
    2. 有能力的人训练那些正在学习的人
    3. 熟练测试那些已经接受过培训的人。
      1. 比较那些能胜任这些学习的人的结果
      2. 比较那些被认为合格的人(偶尔)的结果 识别影响质量或一致性的方法"漂移"问题)
      3. 更改SOP以反映当前的最佳做法。
  2. 试剂
    1.  所有抗体试剂应针对细胞类型重新滴定 您将染色(例如血液,PBMC,BAL,等);使用N≥4)。
    2. 所有过程缓冲液应在室温下,不加温   4  °C ,水浴温度设定在37°C。 使用缓冲区的原因在 室温为(1)避免引起的温度冲击 激活,和(2)保持哺乳动物细胞低于温度at 它们是代谢活跃的。 室温可靠 都。
  3. 细胞
    1. 新鲜收获的细胞产生更一致的结果
      1. 解冻的细胞由于许多原因是可变的
      2. 低温保存时的可变效率
      3. 可变存储时间,质量
      4. DMSO毒性
      5. 可变解冻质量
        当存在DMSO时,保持细胞免于升温至代谢活性
      6. 冻融(F/T)循环产生的破碎的细胞碎片,并粘附在活细胞上
      7. 完整的F/T杀死细胞的尸体看起来像活细胞(和 人为地增加"无应答"的分母总和
      8. 初始细胞在F/T循环期间优先(但可变地)丢失
    2. CFC测定设计为1e6 Ly /管,但也适用于0.5-2e6   Ly /管,在1ml培养物中。 如果你减少培养体积,培养基 枯竭和废物积累在活跃的文化可以不同的影响 结果质量内部运行。
  4. 样品ID,标签
    1. 始终按照合理,可重复,一致应用的顺序排列样品
      1. 例如。我们的猴子ID是5位数字
        随机顺序:25113,25131,23115,25311
        数字排序:23115,25113,25131,25311
        这样,您可以始终确保它们处于逻辑,可重现的顺序
    2. 给示例两个名称,一个是简化的简化,方便用户使用
      1. 例如。 A = 23115,B = 25113,C = 25131,D = 25311
      2. 或者,如果他们都在"A组":A1 = 23115,A2 = 25113,A3 = 25131,A4 = 25331
      3. 这样,操作者可以处理(A,B,C,D)或(A1,A2,A3,A4) 的那些混乱的5位数字(这只是乞求得到 混合)。
      4. 此外,如果数组中的一个数字被错误转录,则可以推断出错误并纠正错误。
    3. 数字识别抗原以类似的方式
      1. 例如 1 = Neg,2 = X,3 = Y,... 7 = SEB
      2. 这样,如果你 按照这个顺序将管子定位在机架中,第一个管子将被处理 总是负控制,最后一个将永远是 抗原最可能导致交叉污染问题
      3. 如上所述,这种双重识别有助于明确纠正转录错误。
      4. 这种双重识别在FlowJo和Excel中有好处 FlowJo   仅识别文件名的前32个字符。 如果32 nd 字符是2 =,您在a中具有该样本的唯一标识 方法,如果你正在列出几个SIV_()抗原,你不会 后   你从FlowJo导出数据,你最终得到一个长的文件名   文本字符串,其中多个离散的信息片段 嵌入式。 Excel工具"文本到数据"对于"爆炸" 文件名组成部分,像'='这样的字符很方便 这个过程。
    4. 使用计算机打印的标签
      1. 使用衬线字体(例如,小写的"L"不与"1"混淆)。
      2. 使用大小足够容易阅读的字体。
      3. 使用理解的缩写,以允许更大的字体大小。
      4. 使用标签颜色来传达分组,时间点,猴子,抗原,等
  5. 处理
    1. 使用管夹架
      'Tube UP'表示尚未发生步骤(例如鸡尾酒加入)。
      'Tube DOWN'表示步骤完成。
    2. 将试剂管横向移动,或者在机架中向后/向前移动,以指示它们是否已被使用
    3. 准备步骤或试剂的清单,并在使用试剂或完成步骤时将其标记出来。
    4. 在管子上使用标签放置,以将它们始终定位在机架中。
      1. 试剂1可以在一面分配。
      2. 试剂2,a   随后的步骤中,可以用不同的壁来中继分配 降低交叉污染的可能性。
    5. 组织样品,以便总是处理阴性对照管   第一,最有效的抗原管总是最后处理。 这使得任何交叉污染的后果最小化
    6. 始终以相同的顺序处理管。 这增加 他们每个过程都将获得类似持续时间的可能性 (例如溶血治疗,抗体暴露,等)。
    7. 制作试剂 鸡尾酒,而不是提供单个剂量。 更大的测量 更准确,而且您所做的测量越少,越少 可能你会犯错误。
    8. 经常拖动微量移液器 当从试剂瓶中取出试管时,向上提起试管的侧面。 那 方式,你拖动吸移管外面的任何流体 小费。 这在测量非常小体积时尤其重要 肽抗原
    9. 移液器在其中间位置是最准确的。 使用P100测量50μl,而不是P200或P50。
    10. 如果可能,设置多个阴性对照,并使用任何背景扣除的平均值。
    11. 如果面对这么多的样品,处理周期也是 长期保持质量,将工作分解成可以做到的部分 高品质。 有时,此问题仅适用于单个步骤。 例如,最可能导致质量问题的CFC步骤是 加入Lyse。 如有必要,一次做分批,以便 保持质量。
  6. 弄脏
    1. 使用经过验证的染色板
    2. 将最亮的荧光团置于最罕见的事件上。
    3. 将最有效的荧光团置于最充足的和/或最强烈的事件上。
    4. 请注意PE-Cy7进入PE通道的光谱泄漏等问题,因此将PE-Cy7置于罕见的调光事件上。
    5. 染色体积很重要,所以使用一致的50μl鸡尾酒量使结果更一致。
  7. 收购
    1. 样品应在72小时内采集
    2. 在我们的经验中,用Alexa-700染色的样品遭受快速的质量侵蚀,并且应该在24小时内获得
    3. 如果血细胞计数器积压或功能障碍意味着一些CFC样品将等待 在采集前72h以上,可以停止染色过程   在Lyse后,细胞内染色延迟到刚才 收购。
  8. 设备
    1. 每件关键设备应分配给一名工作人员, 谁负责监测,维持,绩效评估, 和修复。
    2. 库尔特计数器可靠地只计数血液样本,   并且是众所周知的不准确的任何东西。 他们可以做 巨大的变量,巨大的不准确的计数与碎片丰富的样品 BAL,解冻细胞和酶消化的固体组织
    3. 流式细胞仪:
      1. 血细胞计数器应由BD CST系统(或功能相当的性能监测系统)监测
      2. 激光器应在使用前至少预热45分钟。
      3. 流体应该至少每天清洗一次(再次,如果很多 的"脏"样品,导致积累,改变时间 延迟)。
      4. 补偿:
        使用补偿珠,而不是染色细胞(珠更明亮。) 每周制作新鲜补偿珠样品。
        使   所有抗体试剂的补偿样品(这是特别 对于串联染料是重要的,如PerCP-Cy5.5,PE-TexasRed,PE-Cy7, APC-Cy7,等)。
        补偿每个实验(而不是使用前几周的补偿矩阵)。
      5. 获取速度很重要
        离散   人群可以可靠地容忍比模糊更好的"模糊"。 因此,CFC无CD28 v CD95或Ki67门控可以更快地运行   门控。

食谱

  1. Aqua LIVE/DEAD工具包
    1. 浓缩染料
      1瓶溶解在50μlDMSO中的小瓶粉末,可以冷冻等分试样储存数周
    2. 染色溶液(新鲜)
      1. 将2μl浓染料稀释到78μl去离子水(DI)中
      2. 稀释50微升的步骤5bi的溶液到950微升1×PBS
      3. 使用前保存在冰上
  2. 'R10'组织培养基
    1. RPMI-1640(1x)(w/o L-谷氨酰胺,0.1μm过滤)
    2. 胎牛血清(定义,热灭活,40 nm过滤)
      存储为冻结等分
    3. 青霉素+链霉素(P/S)溶液
      存储为冻结等分
    4. L-谷氨酰胺(200mM) 存储为冷冻等分试样; 解冻时感光
    5. 丙酮酸钠(SP)
    6. β-巯基乙醇(bME)(注40)
      光敏感; 存放在铝箔包装瓶
    7. 无菌过滤装置500ml容量0.22μm纤维素乙酸酯过滤器
    8. 公式
      1. 解冻冻结的组件
      2. 在过滤系统顶部结合添加剂,然后过滤
        50ml FBS
        10ml P/S
        10ml L-谷氨酰胺 5 ml SP
      3. 用无菌RPMI冲洗过滤器
      4. 将另外的RPMI直接倒入滤液中,至500ml
      5. 在旋转R10时,缓慢加入500μlbME
      6. 盖紧; 保护光; 储存于4℃
  3. 'PAB'(磷酸盐白蛋白缓冲液)洗涤缓冲液
    1. DPBS,在1x时为10L,在10x时为1L
    2. 牛血清白蛋白(BSA)
    3. 叠氮化钠(防腐剂; NaN3)
    4. 高毒性(测量时戴防护面具!)
    5. 去离子,过滤水(因为叠氮化物,不需要高压灭菌)
    6. 10x原液的配方:
      1. 将4L去离子水分配到10L的浮筒中
      2. 加入灭菌搅拌棒
      3. 顶部有磁力搅拌器,开始温和的漩涡
      4. 将50g BSA缓慢加入漩涡顶部(避免结块)
      5. 缓慢加入22.75克NaN 3
      6. 在室温下搅拌过夜,
      7. 第二天,所有以前的添加剂溶解后
      8. 加入五瓶粉状DPBS的内容物(每瓶在10x时为1L)
      9. 用碳氢化合物冲洗瓶子,然后返回
      10. 提高搅拌速度; 每2小时检查一次,直到所有溶解
      11. 将10x溶液分配到无菌1L瓶中; 盖紧
      12. 将瓶子保存在4°C
    7. 稀释到1x工作股票
      1. 倒入一个空的和灭菌的20 L碳水化合物,倒两个1瓶10x PAB股票
      2. 用去离子水冲洗倒空的1升瓶子,返回
      3. 加入去离子水至炭黑,直到总体积为20 L
      4. 盖,然后通过反转混合
      5. 储存在4℃的carbuoy
      6. 使用1x工作股票
      7. 使用泵分配器(例如,Brinkmann Bottletop Dispenser;例如,Brand Tech Dispensette)转移到瓶子中   
      8. 在室温4℃或室温下使用PAB
  4. '溶解固定和RBC裂解溶液(注17)
    1. BD FACS裂解溶液,10倍浓缩物
      注意:包含多聚甲醛。
    2. 去离子水
    3. 公式
      1. 按以下比例混合成分
      2. 50 ml 10x FACS裂解液
      3. 450ml去离子水
      4. 通过摇动,搅拌或倒置混合
      5. 分配到深色塑料瓶
      6. 在室温下储存密封的瓶子
      7. QC测试与全血可接受的结果
  5. 'Perm'(1x)固定和不可逆细胞渗透溶液(注18)
    1. 1x"Lyse"(从上面)
    2. 吐温-20(聚氧乙烯脱水山梨糖醇单月桂酸酯)
    3. 这需要高粘度移液器测量良好
    4. 公式
      1. 向1L'Lyse'中加入500μlTween-20,同时搅拌
      2. 在磁力搅拌器上搅拌过夜
      3. 分配到深色塑料瓶
      4. 在室温下存放带盖的瓶子
      5. QC测试与Ki67抗体,为可接受的结果

致谢

本协议中描述的方法自本协议中引用的1995年论文(Picker等人,1995年)以来一直在发展,并且在我们的小组(Hansen等人)发表的两篇2013年论文中使用, et al。,2013a和Hansen等人,2013b)。 NIH和比尔和梅林达·盖茨基金会提供了资金。

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引用:Sylwester, A. W., Hansen, S. G. and Picker, L. J. (2014). Quantification of T Cell Antigen-specific Memory Responses in Rhesus Macaques, Using Cytokine Flow Cytometry (CFC, also Known as ICS and ICCS): from Assay Set-up to Data Acquisition. Bio-protocol 4(8): e1110. DOI: 10.21769/BioProtoc.1110.
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