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Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet
使用Celltrace Violet示踪法进行体外T淋巴细胞增殖和调节性T细胞抑制实验   

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Abstract

Measurement of the incorporation of radionuclides such as 3H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.
FoxP3+ regulatory T cells (Treg) are critical mediators of immune tolerance and evaluation of their functionality is an important step in characterizing many immune models (Rudensky, 2011). Classically CD4+ Treg and conventional or “responder” T cells have been isolated based on their surface expression of CD25 (Treg: CD4+CD25+, Tresp: CD4+CD25-). However, we and others have noted that populations of CD4+CD25- cells express the FoxP3 transcription factor and have suppressive function. Therefore we have utilized the transgenic FoxP3-EGFP mouse to facilitate live purification of suppressor and responder populations based on EGFP (and thus FoxP3) expression. Here we present our adapted protocol for assaying regulatory T cell suppression of Celltrace Violet-labeled responder T cells.

Keywords: Treg(调节性T细胞), Proliferation(增殖), Suppression(抑制), Regulatory t cell(调节 T 细胞), Flow cytometry(流量细胞计数法)

Materials and Reagents

  1. 70 μm nylon mesh cell strainers (Thermo Fisher Scientific, FisherbrandTM, catalog number: 22363548 )
  2. 5 ml polypropylene round-bottom FACS tubes (BD Falcon®, catalog number: 352063 )
    Note: Currently, it is “Corning, catalog number: 352063”.
  3. 50 m conical polypropylene tubes (BD Falcon®, catalog number: 352098 )
    Note: Currently, it is “Corning, catalog number: 352098”.
  4. 96 well plate, U bottom (BD, Falcon®, catalog number: 353077 )
    Note: Currently, it is “Corning, catalog number: 353077”.
  5. Mice
    1. B6-FoxP3EGFP strain (B6.Cg-Foxp3tm2Tch/J) (Jackson Laboratories, catalog number: 006772 ) (see Note 1)
    2. Rag KO (Rag 1 KO, B6.129S7-Rag1tm1Mom/J) (Jackson Laboratories, catalog number: 002216 )
  6. Biosure preservative-free 8x sheath fluid concentrate (for cell sorter) [Cedarlane, catalog number: 1027(BS) ]
  7. Hank’s Balanced Salt solution (HBSS) (Life Technologies, InvitrogenTM, catalog number: 14170 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: 14170”.
  8. Fetal bovine serum (Life Technologies, InvitrogenTM, catalog number: 16170078 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 16170078”.
  9. 1 M HEPES (Life Technologies, InvitrogenTM, catalog number: 15630080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630080”.
  10. PE anti-mouse CD8a (clone 53-6.7) (eBioscience, catalog number: 120081 )
  11. APC anti-mouse CD4 (clone GK1.5) (eBioscience, catalog number: 170041 )
  12. PerCP-eFluor 710 anti-mouse CD4 (clone RM4-5) (eBioscience, catalog number: 460042 )
  13. APC-eFluor 780 anti-mouse TCRβ (clone H57-597) (eBioscience, catalog number: 475961 )
  14. Ultracomp eBeads (eBioscience, catalog number: 012222 )
  15. “Fc blocking” antibody, anti-mouse CD16/CD32 (clone 2.4g2) (9.6 mg/ml) (Bio X cell, catalog number: CUS-HB-197 )
  16. Functional grade purified anti-mouse CD3ε (clone 145-2C11) (eBioscience, catalog number: 160031 )
  17. Celltrace violet cell proliferation kit (Life Technologies, InvitrogenTM, catalog number: C34557 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: 16170078”.
  18. LIVE/DEAD Fixable Yellow Dead cell stain kit (Life Technologies, InvitrogenTM, catalog number: L34959 ) or 7-Aminoactinomycin D (7-AAD) (Sigma-Aldrich, catalog number: A9400)
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L34959”.
  19. 18 MΩ.cm deionized water
  20. Dulbecco modified Eagle medium (DMEM) (Life Technologies, InvitrogenTM, catalog number: 11965 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 11965 ”.
  21. Pen/strep (100 U/ml Penicillin, 100 μg/ml Streptomycin) (Life Technologies, InvitrogenTM, catalog number: 15140122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140122”.
  22. Non-essential amino acids (Life Technologies, InvitrogenTM, catalog number: 11140050 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11140050”.
  23. Sodium pyruvate (Life Technologies, InvitrogenTM, catalog number: 11360070 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11360070”.
  24. L-glutamine (Life Technologies, InvitrogenTM, catalog number: 25030081 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25030081”.
  25. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  26. Enriched DMEM (E-DMEM) (see Recipes)
  27. Phosphate-buffered saline (PBS) (see Recipes)
  28. ACK lysis buffer (see Recipes)

Equipment

  1. Cell sorter (BD Biosciences, model: BD Influx )
  2. Flow cytometer with 405 nm excitation capability (see note 3) (BD Biosciences, model: BD LSR II )
  3. 37 °C humidified incubator, 5% CO2
  4. Centrifuge (capable of spinning 50 ml conical tubes, 5 ml FACS tubes and 96 well plates )
  5. Multichannel pipette (30-300 μl) (Eppendorf)

Procedure

  1. Cell preparation
    Note: All procedures involving animals must be performed according to approved institutional, regional or national protocols (as applicable) by appropriately trained and certified personnel.

    1. Euthanize mice by cervical dislocation and aseptically remove the spleen.
    2. Prepare splenocytes via standard methods. We use firm but gentle disruption of spleens through a 70 μm nylon mesh filter basket with the plunger from a 3 ml syringe, keeping tissue moist and washing cells through with HBSS + 2% FBS at room temperature. For red blood cell lysis, pellet the cells by centrifuging at ~320 x g for 5-10 min @ RT. Resuspend cell pellet in 2-5 ml ACK lysis buffer at room temperature for 5 min with periodic mixing by swirling, followed by adding ~10 volumes (20-50 ml) of HBSS + 2% FBS and spinning at ~320 x g for 5-10 min @ RT.
    3. Resuspend cells in HBSS + 2% FBS at room temperature. Add Fc blocking antibody and wait 5 min. Add APC anti-CD4 and PE anti-CD8 antibodies and incubate at RT for ~30 min. [Typically we resuspend splenocytes from 4 adult mice in 2 ml buffer and use 25 μl Fc block antibody stock (0.24 mg), 25 μl APC anti-CD4 (5 μg), 50 μl PE-anti CD8 (10 μg).]
    4. Spin cells at ~320 x g for 5-10 min @ RT. Wash cells 1x with at least 10 staining volumes (i.e.,: > 20 ml) of HBSS + 2% FBS.
    5. Resuspend splenocytes in sufficient HBSS + 20% FBS + 10 mM HEPES to give a concentration of ~15 x 106 cells/ml. Filter cells through a 70 μm nylon mesh filter and place cells on ice.
    6. Proceed to sort the CD4 single positive (SP) GFP- and CD4 SP GFP+ cell populations, directly into separate 5 ml FACS tubes containing about 1 ml of 99% FBS + 1% 1 M HEPES (final concentration 10 mM). Exclude doublets based on FSC-H vs FSC-A plots. Emphasize purity over yield. Store tubes containing sorted cells on ice after removing from the sorter. We have found it is not necessary to chill the tubes during collection on the sorter.
    7. Evaluate the purity of the sorted cell populations by flow cytometry. Typical yield of CD4+ GFP+ cells from one wild-type mouse spleen after sorting is approximately 7 x 105 cells. Typical yield of CD4+ GFP- cells from one wild-type mouse spleen after sorting is approximately 5 x 106 cells.

  2. Assay setup
    1. Count, spin cells at ~320 x g for 5-10 min @ RT, and resuspend the CD4+ GFP+ (FoxP3+, “suppressors”) in E-DMEM at a concentration of (2 x 106 cells/ml). Place in a humidified 37 °C 5% CO2 incubator with the lid of the tube loose while preparing the responder cells.
    2. Count and wash (spin cells at ~320 x g for 5-10 min @ RT, then resuspend in the solution specified) the CD4+ GFP- (FoxP3-, “responders”) in HBSS or PBS (without serum) twice. Label approximately twice the number of cells you will need for the assay, as it is common to lose a significant proportion of your cells during the Celltrace violet (CTV) labeling process. Set aside an aliquot of cells that are unlabeled (~5 x 105 cells, resuspended in 500 μl of E-DMEM) for use as 2 controls for later flow cytometric analysis (non-CTV stained, vital stain only; and completely unstained; Figure 1 wells E7 and E8 respectively). These cells should be stored at 37 °C in a humidified 5% CO2 incubator-they can be added directly to the appropriate wells of the assay plate now or stored until assay setup in a 15 ml or 50 ml conical tube in the incubator with the cap on loosely. See Note 4 for discussion of the use of non-fractionated splenocytes as responders.
    3. Label the CD4+ GFP- cells with CTV. Briefly, prepare a 5 mM CTV stock solution according to the manufacturer’s directions by adding 20 μl of the supplied (room temperature) DMSO to an aliquot of dry CTV dye and mixing thoroughly. Prepare a 5 μM working solution by diluting stock 1/1,000 in prewarmed (37 °C) HBSS or PBS. Resuspend pelleted, washed CD4+GFP-cells in the working solution at a concentration of between 1-15 x 106 cells/ml, ensuring complete and rapid cell pellet disruption/ re-suspension by triturating through a serological pipette. Incubate for 20 min in a 37 °C water bath.
    4. Quench the labeling reaction by adding 5 volumes of cold DMEM + 10% FBS. Incubate for 5 min at RT, then wash cells 1x with E-DMEM and resuspend in prewarmed (37 °C) E-DMEM. Incubate at 37 °C in a humidified 5% CO2 incubator for at least 15 min.
    5. Wash cells with 25-50 ml warmed E-DMEM, count and resuspend at 1 x 106 cells/ml in E-DMEM.
    6. While performing the labeling reaction, prepare antigen presenting cells (APC) for the plate assay by euthanizing a Rag KO mouse, removing the spleen, and preparing splenocytes as previous with RBC lysis. Wash, count, and resuspend these cells at 2 x 106 cells/ml in E-DMEM.
    7. Set up assay plate (see Table 1 and Figure 1):
      Each well used of the 96 well U-bottom assay plate should contain:
      1. 1 x 105 Responder T cells
      2. Either 1 x 105 (Treg: Tresp ratio 1:1), 5 x 104 (1:2), 2.5 x 104 (1:4), 1.25 x 104 (1:8), 6.25 x 103 (1:16) or 0 (0:1) suppressor T cells
      3. 1 μg/ml anti-CD3ε antibody
      4. 1 x 105 Antigen presenting cells (RBC-depleted splenocytes from Rag KO animal(s), prepared in step B13 above)
      5. Sufficient E-DMEM media to give a final volume of 250 μl per well.
        Notes: Each condition should be assayed at least in triplicate. Try to avoid using the edge wells of the assay plate as they are subject to increased evaporation rates. Remember to include 2 sets of wells containing no Treg and no anti-CD3ε stimulation: One set will serve as the unstimulated control (Figure 1, wells B6-B9), the other will be used as a compensation control for the CTV stain during later flow cytometric analysis (Figure 1, wells D7-D9). Add the non-CTV labeled cells saved from step 9 to two wells of the assay plate (Figure 1, wells E7-E8).

        Table 1. Suppression assay set up guide-each well contains the following volumes of components for each condition in the left column



        Figure 1. Example plate layout for a typical suppression assay with experimental wells set up in quadruplicate. Ratios are expressed as Treg: Tresponders.

    8. Incubate the assay plate in a 37 °C, 5% CO2 humidified incubator for 5 d.

  3. Staining
    1. Centrifuge the assay plate for 5 min at ~400 x g.
    2. Remove the supernatant from the wells with a quick “flicking” motion into an approved receptacle.
    3. Using a multichannel pipette, add 200 μl HBSS with no serum (if labeling cells with fixable yellow live/dead stain) or 200 μl HBSS + 2% FBS (if using 7-AAD for dead cell discrimination) to each well to wash. Pipet up and down ~3 times to resuspend the cells. Centrifuge the assay plate again for 5 min at ~400 x g and flick off the supernatant.
      Note: If you are using fixable yellow live/dead stain in your assay perform steps 19-22, otherwise skip to step 23.
    4. Prepare sufficient fixable yellow live/dead stain in HBSS according to the manufacturer’s protocol such that 200 μl is available per well of the plate that was used. Briefly, 50 μl of the supplied DMSO is added to one vial of the dry dye solution and mixed. This reconstituted dye solution is then diluted 1/1,000 in HBSS for use in labeling.
    5. Resuspend cells in each well (excluding the CTV-only control wells and unstained control wells, Figure 1 wells D7-D9 and E8) in 200 μl LIVE/DEAD fixable yellow stain/ HBSS by pipetting up and down with a multichannel pipette. Store plate on ice in the dark for 30 min.
    6. Centrifuge the plate for 5 min at ~400 x g, remove supernatant, and wash with 200 μl/well HBSS + 2% FBS.
    7. Centrifuge the plate for 5 min at ~400 x g, remove supernatant.
    8. Proceed with staining in the plate using your desired panel of antibodies with or without an Fc blocking step.

  4. Example staining
    1. Add 30 μl Fc block cocktail to each experimental (non-compensation control) well (1 μl Fc block antibody stock, 29 μl HBSS + 2% FBS, final 9.6 μg of Fc block per well). Pipet up and down to mix. Incubate at RT for 5 min. For larger experiments it is handy to do this with a multichannel pipette by preparing the cocktail and distributing it across several wells of a source 96 well plate.
    2. Without washing, add 30 μl surface marker staining cocktail per experimental well and pipet up and down to mix. Incubate at room temperature for ~30 min.
      Example surface staining cocktail (per staining, all Abs purchased from eBioscience):
      1 μl PE anti-mouse CD8a (0.2 μg)
      0.25 μl PerCP-eFluor 710 anti-mouse CD4 (0.05 μg)
      0.75 μl APC-eFluor 780 anti-mouse TCRβ (0.15 μg)
      28 μl HBSS + 2% FBS
    3. While the cells are incubating, create single-stained compensation controls for each antibody used in your flow cytometry experiment using Ultracomp eBeads in 5 ml round bottom FACS tubes according to the manufacturer’s protocols.
    4. Wash wells 2x with HBSS + 2% FBS. Resuspend in 200 μl per well HBSS + 2% FBS for analysis. If using 7-AAD for dead cell discrimination, resuspend all wells except for the CTV control and unstained control wells in 200 μl per well HBSS + 2% FBS + 1 μg/ml 7-AAD. (Resuspend the CTV control and unstained control wells in 200 μl HBSS + 2% FBS with no 7-AAD.)
    5. Analyze on flow cytometer. For each well we typically remove the 200 μl per well from the plate, transfer it to a 5 ml round-bottom FACS tube containing 400 μl HBSS + 2% FBS, and load on the cytometer for acquisition. We generally analyze cells without fixation, however one could fix cells with ~1% formaldehyde in PBS prior to analysis if desired or required by the local flow cytometry facility. Please note that fixation is not compatible with using 7-AAD for dead cell discrimination, and will generally destroy GFP fluorescence in GFP+ cells.

  5. Analysis tips and sample data
    We suggest drawing a gate containing the lymphocytes based on forward and side scatter (FSC/SSC), and if dead cell discrimination is desired, gating the live cells (i.e.,: LIVE/DEAD fixable yellow stain negative/low population, or 7-AAD low population, see Figures 2A, B respectively). The unstimulated wells will have a clear population of dead cells to help draw the dead cell gate. Then gate the TCRβ+, CD4 SP cells. We have noted that using anti-CD3ε (clone 145-2C11) for stimulation decreases staining intensity for TCRβ (using the H57-597 clone), keep this in mind when drawing gates for TCRβ+, CD4 SP cells in the stimulated and unstimulated conditions.
    Next, in order to analyze the extent of proliferation, examine this gated TCRβ+, CD4 SP population for CTV (Figure 2). Wells that contain Treg (which are not labeled with CTV) will have a population of TCRβ+CD4+ cells that were not stained with CTV and must be excluded from the analysis-use the unstained sample along with the 1:1 Treg:Tresp samples (which should show low responder proliferation allowing easy delineation of Treg vs responder populations) to determine the threshold to place a gate encompassing all CTV-positive events (see Figure 2C for clarification). One cannot exclude Treg on the basis of FoxP3 positivity as some Treg may have lost FoxP3 expression during the assay. Then use the unstimulated samples (i.e.,: Figure 1, wells B6-B9) to draw a gate encompassing all CTV-positive events that have proliferated at least once (Figure 2D). If the assay has incubated for too long, some extensively proliferated cells may have lost enough dye to become indistinguishable from CTV-negative cells. In this case we suggest shortening your assay incubation time to avoid this issue. We are able to resolve 7 generations of cell division and thus we report the data as the percent of generation 0 to 7 cells that have proliferated at least once. It is also important to present the proliferation data visually, as in Figure 2E.


    Figure 2. Analysis of Celltrace Violet staining following suppression assay incubation and sample data. A. Example of gating live cells for analysis based on negative/low staining with LIVE/DEAD yellow fixable stain or B. 7-AAD. C. Examining a sample containing a high ratio of Treg to responder T cells facilitates placement of a gate encompassing true CTV labeled responders and avoiding the unlabeled Treg in the assay. D. Example of gating of CTV+ cells from generations 0-7 that have proliferated at least once. E. Visual comparison of the extent of responder T cell proliferation using various ratios of Treg to responder T cells.

Notes

  1. The FoxP3-EGFP strain used for these studies (B6.Cg-Foxp3tm2Tch/J) has been engineered to produce a bicistronic transcript from the endogenous foxp3 locus encoding the normal endogenous FoxP3 protein as well as EGFP. Another strain referred to by the same common “FoxP3-EGFP” name instead encodes a FoxP3-EGFP fusion protein at the endogenous foxp3 locus. This fusion protein has been shown to have altered function and we thus cannot recommend its use. The fusion protein strain contains the Foxp3 allele, developed by the Rudensky group, and was previously available through The Jackson Laboratory although it has since been removed from their catalog. Please see Chatila and Williams (2012) and references therein for a more comprehensive overview of the issue.
  2. While we do not use this approach, the assay should be easily amenable to the use of Treg and responders purified based on the classical CD4+CD25+/CD4+CD25- surface expression phenotype.
  3. The flow cytometer used in our facility is configured to detect Celltrace violet with a 450 nm/50 nm bandpass filter set, and LIVE/DEAD fixable yellow stain with a 475 low-pass filter + 525 nm/50 nm bandpass filter set. One could substitute other vital stains instead of the LIVE/DEAD fixable yellow stain or 7-AAD as required to be detectable with one’s particular cytometer configuration/requirement for fixation.
  4. Instead of using CD4+FoxP3- cells as responders, one can also substitute Celltrace-violet labeled, unfractionated splenocytes as responder cells in this assay. For this approach we use 2 x 105 labeled splenocytes per well as responders, and omit adding Rag KO splenocytes as APC.

Recipes

  1. Enriched DMEM (E-DMEM)
    10% fetal bovine serum
    1% Pen/strep (100 U/ml Penicillin, 100 μg/ml Streptomycin)
    1% non essential amino acids
    1% sodium pyruvate
    2 mM L-glutamine
    50 μM 2-mercaptoethanol
  2. Phosphate-buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    10mM Na2HPO4
    1.8 mM KH2PO4
  3. ACK lysis buffer
    15.5 mM NH4Cl
    1 mM KHCO3
    0.01 mM EDTA

Acknowledgments

The authors would like to thank Perveen Anwar for assistance with animal care. All animal work described herein was undertaken in accordance with approved animal care protocols at the University of Alberta. This work was supported by a grant from CIHR (FRN79521) to CCA and by doctoral studentships from the Alberta Diabetes Institute and Alberta Innovates Health Solutions (AIHS) to KKE and a senior scholar award from AIHS to CCA. This work was also supported by an Alberta endMS Regional Research and Training Centre Graduate Student Support Grant of the endMS Research and Training Network from the Multiple Sclerosis Society of Canada and the Multiple Sclerosis Scientific Research Foundation.

References

  1. Chatila, T. A. and Williams, C. B. (2012). Foxp3: shades of tolerance. Immunity 36(5): 693-694.
  2. Ellestad, K. K., Thangavelu, G., Ewen, C. L., Boon, L. and Anderson, C. C. (2014). PD-1 is not required for natural or peripherally induced regulatory T cells: Severe autoimmunity despite normal production of regulatory T cells. Eur J Immunol 44(12): 3560-3572.
  3. Kruisbeek, A. M., Shevach, E. and Thornton, A. M. (2004). Proliferative assays for T cell function. Curr Protoc Immunol Chapter 3: Unit 3 12.
  4. Rudensky, A. Y. (2011). Regulatory T cells and Foxp3. Immunol Rev 241(1): 260-268.

简介

放射性核素如3 H胸腺嘧啶核苷的掺入的测量是用于测定T细胞增殖的经典免疫学技术。然而,这种方法具有超出使用放射性材料的不便之处的缺点,例如体放射性核素结合测量不能准确地定量T细胞分裂,以及不能结合增殖分析与同时评价分裂的细胞标记物的表达细胞。通过用诸如CFSE,Celltrace Violet等活性染料标记T细胞,在每个细胞分裂的子细胞之间平均分配T细胞,可以通过流式细胞术相对容易地追踪增殖细胞的代和它们的各种分子的表达。 > FoxP3 + 调节性T细胞(Treg)是免疫耐受的关键介质,其功能评价是表征许多免疫模型的重要步骤(Rudensky,2011)。基于其表面表达的CD25(Treg:CD4 + CD25 + sup/+),已经分离了经典的CD4 + Treg和常规或"应答者"T细胞, ,Tresp:CD4 + CD25 - sup/- )。然而,我们和其他人已经注意到,CD4 + CD25 - 细胞群表达FoxP3转录因子并具有抑制功能。因此,我们利用转基因FoxP3-EGFP小鼠促进基于EGFP(并因此FoxP3)表达的抑制基因和应答者群体的活性纯化。在这里我们提出我们适应的协议,用于测定调节性T细胞抑制Celltrace紫罗兰标记响应T细胞。

关键字:调节性T细胞, 增殖, 抑制, 调节 T 细胞, 流量细胞计数法

材料和试剂

  1. 70μm尼龙网孔细胞过滤器(Thermo Fisher Scientific,Fisherbrand TM ,目录号:22363548)
  2. 5ml聚丙烯圆底FACS管(BD Falcon ,目录号:352063)
    注意:目前,它是"康宁,目录号:352063"。
  3. 50μm圆锥形聚丙烯管(BD Falcon,目录号:352098) 注意:目前,它是"康宁,目录号:352098"。
  4. 96孔板,U底(BD,Falcon ,目录号:353077)
    注意:目前,它是"康宁,目录号:353077"。
  5. 小鼠
    1. (Jackson Laboratories,目录号:006772)(参见注释1)的B6-FoxP3 EGFP 株(B6.Cg-Foxp3 tm2Tch
    2. Rag KO(Rag 1 KO,B6.129S7-Rag 1tm1Mom )(Jackson Laboratories,目录号:002216)
  6. 无生物防腐剂的8x鞘液浓缩液(细胞分选机)[Cedarlane,目录号:1027(BS)]
  7. Hank's平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM ,目录号:14170)
    注意:目前,它是"Thermo Fisher Scientific,目录号:14170"。
  8. 胎牛血清(Life Technologies,Invitrogen TM ,目录号:16170078)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:16170078" />
  9. 1 M HEPES(Life Technologies,Invitrogen TM ,目录号:15630080)
    注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:15630080" />
  10. PE抗小鼠CD8a(克隆53-6.7)(eBioscience,目录号:120081)
  11. APC抗小鼠CD4(克隆GK1.5)(eBioscience,目录号:170041)
  12. PerCP-eFluor 710抗小鼠CD4(克隆RM4-5)(eBioscience,目录号:460042)
  13. APC-eFluor 780抗小鼠TCRβ(克隆H57-597)(eBioscience,目录号:475961)
  14. Ultracomp eBeads(eBioscience,目录号:012222)
  15. "Fc阻断"抗体,抗小鼠CD16/CD32(克隆2.4g2)(9.6mg/ml)(Bio X cell,目录号:CUS-HB-197)
  16. 功能级纯化的抗小鼠CD3ε(克隆145-2C11)(eBioscience,目录号:160031)
  17. Celltrace紫细胞增殖试剂盒(Life Technologies,Invitrogen TM ,目录号:C34557)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:16170078" br />
  18. LIVE/DEAD可固定黄色死细胞染色试剂盒(Life Technologies,Invitrogen ,目录号:L34959)或7-氨基放线菌素D(7-AAD)(Sigma-Aldrich,目录号:A9400) /> 注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号:L34959" br />
  19. 18MΩ cm去离子水
  20. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Invitrogen TM ,目录号:11965)
    注意:目前,"赛默飞世尔科技有限公司 ,目录号:11965" />
  21. Pen/strep(100U/ml青霉素,100μg/ml链霉素)(Life Technologies,Invitrogen TM,目录号:15140122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140122" />
  22. 非必需氨基酸(Life Technologies,Invitrogen TM,目录号:11140050)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11140050" />
  23. 丙酮酸钠(Life Technologies,Invitrogen TM,目录号:11360070) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11360070" />
  24. L-谷氨酰胺(Life Technologies,Invitrogen TM,目录号:25030081) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25030081" />
  25. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  26. 富含DMEM(E-DMEM)(参见配方)
  27. 磷酸盐缓冲盐水(PBS)(见配方)
  28. ACK裂解缓冲液(见配方)

设备

  1. 细胞分选仪(BD Biosciences,型号:BD Influx)
  2. 具有405nm激发能力的流式细胞仪(参见注释3)(BD Biosciences,型号:BD LSR II)
  3. 37℃湿润培养箱,5%CO 2/h
  4. 离心机(能够旋转50ml锥形管,5ml FACS管和96孔板)
  5. 多通道移液器(30-300μl)(Eppendorf)

程序

  1. 单元格准备
    注意:涉及动物的所有程序必须根据经过适当培训和认证的人员的认可机构,地区或国家协议(如适用)进行。

    1. 安乐死小鼠颈椎脱位和无菌取出脾
    2. 通过标准方法准备脾细胞。我们使用坚定但温和 通过70微米的尼龙网过滤篮破坏脾脏 柱塞从3ml注射器,保持组织潮湿和洗涤细胞 通过HBSS + 2%FBS。对于红细胞 裂解,通过在?320×g离心5-10分钟在RT下沉淀细胞。 重悬细胞沉淀在2-5毫升ACK裂解缓冲液在室温下 ?5分钟,通过涡旋进行周期性混合,然后加入?10体积 (20-50ml)HBSS + 2%FBS,并在?320×g下旋转5-10分钟,室温。
    3. 重悬细胞在HBSS + 2%FBS室温。添加Fc 封闭抗体,并等待5分钟。添加APC抗CD4和PE抗CD8 抗体并在室温下孵育?30分钟。 [通常我们重悬 来自4只成年小鼠的脾细胞在2ml缓冲液中,并使用25μlFc块 抗体储液(0.24mg),25μlAPC抗CD4(5μg),50μlPE-抗CD8 (10μg)。]
    4. 在?320×g下旋转细胞5-10分钟,室温。洗涤细胞1x ?与至少10个染色体积(即,:> 20ml)的HBSS + 2%FBS。
    5. 在足够的HBSS + 20%FBS + 10mM HEPES中重悬脾细胞 得到?15×10 6个细胞/ml的浓度。过滤细胞通过70μm ?尼龙网过滤器,将细胞置于冰上
    6. 继续排序 CD4单阳性(SP)GFP和CD4 SP GFP +细胞群体 ?分入含有约1ml的99%FBS + 1%1M的单独的5ml FACS管中 ?HEPES(终浓度10mM)。排除基于FSC-H vs的双峰 FSC-A图。强调纯度高于产量。存储管排序 从分选机中取出后,冰上的细胞。我们发现它不是 必须在分拣机上收集时冷却试管
    7. 通过流式细胞术评估分选的细胞群体的纯度。 来自一个野生型小鼠脾脏的CD4 +细胞的典型产量 分选大约是7×10 5个细胞。 CD4 +细胞的典型产量 ?从分选后的一个野生型小鼠脾脏约5×10 6个细胞

  2. 测试设置
    1. 计数,在?320×10 5细胞/cm旋转细胞5-10分钟,并重悬CD4 + GFP + (FoxP3 < (2×10 6个细胞/ml)的浓度的E-DMEM中的"抑制剂"("抑制剂")。置于加湿的37℃,5%CO 2培养箱中 管准备响应细胞时松动。
    2. 计数和 洗涤(在?320×g离心细胞5-10分钟,然后在室温下重悬) 溶液指定)在HBSS或PBS中的CD4 + GFP - (FoxP3 - ,"应答者") (无血清)两次。标签大约是你的单元格数量的两倍 ?将需要测定,因为它是常见的失去一个显着 在Celltrace紫色(CTV)标记过程中您的细胞比例 处理。取出未标记的细胞(约5×10 5个细胞,重悬于500μl的E-DMEM)的等分试样,用作2个对照用于随后 流式细胞术分析(非CTV染色,仅有活体染色; 完全未染色;图1分别为孔E7和E8)。这些 细胞应该在37℃下在潮湿的5%CO 2培养箱中储存 可以直接加入到测定板的适当孔中 ?储存直至在15ml或50ml锥形管中的测定装置 孵育器与帽子松散。有关使用的讨论见注4 的非分级脾细胞作为应答者。
    3. 用CTV标记CD4 + GFP - 细胞。简言之,制备5mM CTV储备溶液 根据制造商的说明加入20μl的 供应(室温)DMSO至干燥CTV染料的等分试样并混合 ?彻底。通过稀释储备1/1,000制备5μM工作溶液 在预热(37℃)HBSS或PBS中。重悬浮沉淀,洗涤 CD4 + sup/+细胞在工作溶液中的浓度为1-15 ?x 10 6个细胞/ml,确保完全和快速的细胞沉淀破坏/ 通过血清移液管研磨重悬浮。孵化为 ?在37℃水浴中20分钟。
    4. 猝灭标记反应 加入5倍体积的冷DMEM + 10%FBS。然后在RT孵育5分钟 用E-DMEM洗涤细胞1×并重悬于预热的(37℃)E-DMEM中。 在37℃在潮湿的5%CO 2培养箱中孵育至少15分钟。
    5. 用25-50ml温热的E-DMEM洗涤细胞,计数并在E-DMEM中以1×10 6个细胞/ml重悬。
    6. 在进行标记反应时,制备抗原呈递 细胞(APC),通过对Rag KO小鼠实施安乐死,去除 脾,并如前所述用RBC裂解制备脾细胞。洗, 计数,并将这些细胞以2×10 6个细胞/ml重悬于E-DMEM中。
    7. 设置测定板(见表1和图1):
      96孔U型底部测定板中使用的每个孔应包含:
      1. 1 x 10 5 响应T细胞
      2. 1×10 5(Treg:Tresp比率1:1),5×10 4 sup(1:2),2.5×10 4 sup/(1:4),1.25×10 4(1:8),6.25×10 3(1:16)或0(0:1)抑制T细胞, br />
      3. 1μg/ml抗CD3ε抗体
      4. 1×10 5抗原呈递细胞(来自上述步骤B13中制备的Rag KO动物的RBC缺乏的脾细胞)
      5. 充足的E-DMEM培养基,使每孔最终体积为250μl 注意:每个条件应至少一式三份进行测定。尝试 避免使用它们受到的测定板的边缘孔 增加蒸发速率。记住包括2套井 不含Treg和没有抗CD3ε刺激:一组将作为 未刺激的对照(图1,孔B6-B9),另一个将被使用 ?作为在后期流动期间CTV污迹的补偿控制 细胞计数分析(图1,孔D7-D9)。添加标记的非CTV 从步骤9保存的细胞至测定板的两个孔(图1,孔 ?E7-E8)。

        表1.抑制测定设置指南 - 每个孔 包含以下卷中的每个条件的组件 左栏



        图1.典型抑制的示例板布局 ?实验设置为一式四份。比率是 表示为Treg:Tresponders。

    8. 孵育测定板在37℃,5%CO 2加湿培养箱中5天。

  3. 染色
    1. 在约400×g离心试验板5分钟。
    2. 通过快速"轻拂"运动,将孔中的上清液移至批准的容器中
    3. 使用多通道移液器,加入200微升没有血清的HBSS(如果 用可固定的黄色活/死染色标记细胞)或200μlHBSS + 2% FBS(如果使用7-AAD进行死细胞鉴别)到每个孔中洗涤。 吸取上下?3次以重悬细胞。离心该试验 在?400×g下再次平板5分钟,并轻轻地离开上清液 注意:如果您在测定中使用可固定的黄色活/死染色,请执行步骤19-22,否则跳至步骤23.
    4. 根据HBSS准备足够的可固定黄色活/死染色 ?到制造商的协议,使得每孔200μl 的板。简言之,加入50μl所提供的DMSO ?加入到一个小瓶的干燥染料溶液中并混合。这种重构染料 溶液在HBSS中稀释1/1000,用于标记
    5. 重悬细胞在每个井(不包括CTV只有控制井和 未染色的对照孔,图1孔D7-D9和E8) LIVE/DEAD可固定黄色染色/HBSS通过上下吹吸a 多通道移液器。将板在冰上在黑暗中储存30分钟。
    6. 在?400×g离心板5分钟,除去上清液,用200μl/孔HBSS + 2%FBS洗涤。
    7. 在?400×g离心板5分钟,除去上清液。
    8. 继续在板中使用您想要的抗体组进行染色,有或没有Fc阻断步骤。

  4. 示例染色
    1. 加入30μlFc块混合物到每个实验(非补偿 对照)孔(1μlFc封闭抗体原液,29μlHBSS + 2%FBS,最终 每孔9.6μgFc嵌段)。吸移上下混合。在RT孵育 5分钟。对于较大的实验,它是方便的做到这一点 多通道移液器通过准备鸡尾酒和分发它 跨越源96孔板的几个孔。
    2. 没有 洗涤,每个实验加入30μl表面标志物染色混合物 井和移液器上下混合。在室温下孵育?30 min。
      实施例表面染色混合物(每次染色,所有购自eBioscience的Ab):
      1μlPE抗小鼠CD8a(0.2μg)
      0.25μlPerCP-eFluor 710抗小鼠CD4(0.05μg)
      0.75μlAPC-eFluor 780抗小鼠TCRβ(0.15μg)
      28μlHBSS + 2%FBS
    3. 当细胞孵育时,创建单染色补偿 对于在使用的流式细胞术实验中使用的每种抗体的对照 Ultracomp eBeads在5ml圆底FACS管中, 制造商的协议。
    4. 用HBSS + 2%FBS洗涤孔2次。 重悬于200微升每孔HBSS + 2%FBS用于分析。如果使用7-AAD 对于死细胞鉴别,重悬所有孔,除了CTV 对照和未染色的对照孔,每孔200μlHBSS + 2%FBS + 1 ?μg/ml 7-AAD。 (重悬CTV对照和未染色的对照孔 200μlHBSS + 2%FBS,不含7-AAD。)
    5. 流式细胞仪分析。 对于每个孔,我们通常从板中移除每孔200μl, 转移到含有400μlHBSS + 2%胎牛血清的5ml圆底FACS管中, ?FBS,并加载到细胞仪上进行采集。我们一般分析 细胞无固定,但可以固定细胞与?1% 甲醛在PBS中分析前如果需要或需要 局部流式细胞术设施。请注意,固定不是 兼容使用7-AAD进行死细胞鉴别,并将 通常破坏GFP + 细胞中的GFP荧光。

  5. 分析提示和样品数据
    我们建议基于正向和侧向散射(FSC/SSC)绘制包含淋巴细胞的门,并且如果需要死亡细胞分化,则选择活细胞(:LIVE/DEAD可固定黄色染色阴性/低人群或7-AAD低人群,分别参见图2A,B)。未刺激的孔将具有清楚的死细胞群体,以帮助绘制死细胞门。然后选择TCRβ + ,CD4 SP细胞。我们已经注意到,使用抗CD3ε(克隆145-2C11)用于刺激降低了TCRβ的染色强度(使用H57-597克隆),在绘制TCRβ + ,CD4SP细胞在刺激和未刺激的条件。
    接下来,为了分析增殖的程度,检查该门控TCRβ + ,CTV的CD4 SP群体(图2)。含有Treg(其未用CTV标记)的孔将具有未被CTV染色的TCRβ+细胞群,并且必须从分析中排除 - 使用未染色的样品连同1:1 Treg:Tresp样品(其应当显示低反应者增殖允许容易地描绘Treg与应答者群体),以确定放置包含所有CTV阳性事件的门的阈值(参见图2C,澄清)。人们不能在FoxP3阳性的基础上排除Treg,因为一些Treg可能在测定期间丧失FoxP3表达。然后使用未刺激的样品(即:图1,孔B6-B9)画出包含已增殖至少一次的所有CTV阳性事件的门(图2D)。如果测定已经孵育太长时间,一些大量增殖的细胞可能已经失去足够的染料,变得不能与CTV阴性细胞区分。在这种情况下,我们建议缩短您的测定孵育时间,以避免这个问题。我们能够解决7代细胞分裂,因此我们报告数据为至少增殖一次的第0代至第7代细胞的百分比。如图2E所示,可视地呈现增殖数据也是重要的。


    图2.在抑制测定孵育和样品数据之后的Celltrace紫染色分析。A基于使用LIVE/DEAD黄色可定影染色剂或B.7D细胞的阴性/低染色进行分析的门控活细胞的实例。 AAD。 C.检测含有高比例的Treg与应答T细胞的样品促进包含真实CTV标记的应答者的门的放置,并在测定中避免未标记的Treg。 D.来自已经增殖至少一次的0-7代的CTV + 细胞的门控的实施例。 E.使用不同比例的Treg与应答T细胞的应答T细胞增殖的程度的视觉比较

笔记

  1. 用于这些研究的FoxP3-EGFP菌株(B6.Cg-Foxp3 tm2Tch /J)已经被工程化以从编码正常内源的内源foxp3基因座产生双顺反子转录物FoxP3蛋白以及EGFP。由相同的共同的"FoxP3-EGFP"名称提到的另一个菌株在内源性foxp3基因座处代替编码FoxP3-EGFP融合蛋白。这种融合蛋白已经显示具有改变的功能,因此我们不能推荐它的使用。融合蛋白菌株含有由Rudensky集团开发的Foxp3 等位基因,并且以前可通过The Jackson Laboratory获得,尽管它已从其目录中去除。请参阅Chatila和Williams(2012年)及其中的参考资料,以更全面地概述这一问题。
  2. 尽管我们不使用这种方法,但是所述测定应当容易使用基于经典CD4 + CD25 + sup/+/CD4 + sup/+细胞的Treg和响应者的纯化, CD25 - 表面表达表型
  3. 我们的设施中使用的流式细胞仪被配置为检测具有450nm/50nm带通滤波器组的Celltrace紫色,以及具有475低通滤波器+ 525nm/50nm带通滤波器组的LIVE/DEAD可固定黄色染色。人们可以替代其他重要的污渍,而不是LIVE/DEAD可固定的黄色染色或7-AAD根据需要可检测与特定的细胞仪配置/要求固定。
  4. 代替使用CD4 + FoxP3 - 细胞作为反应者,也可以在该测定中代替Celltrace-violet标记的未分级的脾细胞作为反应者细胞。对于这种方法,我们使用每孔2×10 5个标记的脾细胞作为应答者,并省略将Rag KO脾细胞作为APC添加。

食谱

  1. 富含DMEM(E-DMEM)
    10%胎牛血清 1%Pen/strep(100U/ml青霉素,100μg/ml链霉素) 1%非必需氨基酸
    1%丙酮酸钠 2mM L-谷氨酰胺 50μM2-巯基乙醇
  2. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4 sub/
  3. ACK裂解缓冲液
    15.5mM NH 4 Cl
    1mM KHCO 3
    0.01mM EDTA

致谢

作者感谢Perveen Anwar对动物护理的援助。本文所述的所有动物工作根据在Alberta大学批准的动物护理方案进行。这项工作得到CIHR(FRN79521)授予CCA和阿尔伯塔糖尿病研究所的博士生奖学金和艾伯塔省创新健康解决方案(AIHS)授予KKE以及AIHS至CCA的高级学者奖。这项工作还得到了来自加拿大多发性硬化症协会和多发性硬化症科学研究基金会的endMS地区研究和培训中心研究生支持资助。

参考文献

  1. Chatila,T.A。和Williams,C.B。(2012)。 Foxp3:宽容的阴影。 免疫力 36(5 ):693-694。
  2. Ellestad,K.K.,Thangavelu,G.,Ewen,C.L.,Boon,L.and Anderson,C.C。(2014)。 PD-1不是天然或外周诱导的调节性T细胞所必需的:严重的自身免疫,尽管正常生产调节性T细胞。 Eur J Immunol 44(12):3560-3572。
  3. Kruisbeek,A.M.,Shevach,E。和Thornton,A.M。(2004)。 T细胞功能的增殖测定。 Curr Protoc Immunol 第3章:第3单元12.
  4. Rudensky,A.Y。(2011)。 Regulatory T cells and Foxp3。 Immunol Rev 241( 1):260-268。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ellestad, K. K. and Anderson, C. C. (2016). Protocol-In vitro T Cell Proliferation and Treg Suppression Assay with Celltrace Violet. Bio-protocol 6(1): e1698. DOI: 10.21769/BioProtoc.1698.
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