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Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)
人单核细胞衍生的树突状细胞(MDDC)捕获HIV-1和葡聚糖的定量测定   

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Abstract

The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

Background

This protocol was developed in order to assess the changes of HIV-1 internalization upon disruption of actin nucleation in human monocyte derived dendritic cells. Following a shRNA screen to identify genes important for HIV-1 transfer from dendritic cells to T cells, we observed that a disruption of actin nucleation leads to a switch from actin rich dendrites to blebs, due to an excess of actomyosin contraction. As a consequence, a decrease of HIV-1 transfer and an increase of HIV-1 internalization due to bleb retraction-driven macropinocytosis were observed. We concluded that effectors of actin nucleation and stabilization were key to maintain HIV-1 on actin-rich dendrites and to limit its endocytosis, for efficient transfer to T lymphocytes (Menager and Littman, 2016).

Materials and Reagents

  1. Production of HIV-1 particles
    1. 10 cm plate (100 x 20 mm) (Corning, Falcon®, catalog number: 353003 )
    2. 15 cm plate (150 x 20 mm) (Corning, Falcon®, catalog number: 353025 )
    3. 15 ml conical tube
    4. 33 mm Millex-HV syringe filter, PVDF, 0.45 μm (EMD Millipore, catalog number: SLHV033RS )
    5. 60 ml syringe with BD Luer-LokTM tip (BD, Luer-LokTM, catalog number: 309653 )
    6. 50 ml high clarity PP centrifuge tube, conical bottom, sterile, 25/bag, 500/case (Corning, Falcon®, catalog number: 352070 )
    7. 293FT cells (Thermo Fisher Scientific, InvitrogenTM, catalog number: R700-07 )
    8. X4-HIV-Gag-iGFP plasmid
      Note: Derived from HIV-1 molecular clone NL4-3 modified to insert the GFP protein in between the MA and CA domains of Gag as previously described (Hubner et al., 2007).
    9. Dulbecco’s modification of Eagles medium (DMEM) (Mediatech, catalog number: 10-017-CV )
    10. Fetal bovine serum (FBS)
    11. MEM amino acid solution (GE Healthcare, HyCloneTM, catalog number: SH30598.01 )
    12. HyCloneTM L-glutamine (GE Healthcare, HyCloneTM, catalog number: SH3003401 )
    13. HyCloneTM penicillin streptomycin 100x solution (GE Healthcare, HyCloneTM, catalog number: SV30010 )
    14. Gentamicin (50 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
    15. Geneticin® selective antibiotic (G418 sulfate) (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 11811023 )
    16. Trypsin 0.05% protease solution with porcine trypsin, HBSS, EDTA; without calcium, magnesium (GE Healthcare, HyCloneTM, catalog number: SH30236.01 )
    17. ProFection® mammalian transfection system-calcium phosphate (Promega, catalog number: E1200 )
    18. Calcium chloride (CaCl2)
    19. 100 mM sodium pyruvate solution (GE Healthcare, HyCloneTM, catalog number: SH30239.01 )
    20. 1 M HEPES solution (GE Healthcare, HyCloneTM, catalog number: SH30237.01 )
    21. D10 medium (see Recipes)

  2. Differentiation of monocyte derived dendritic cells (MDDCs)
    1. 60 ml syringe with BD Luer-LokTM tip (BD, Luer-LokTM, catalog number: 309653 )
    2. Needle (50/sp, 500/ca), 18 G x 1 ½ in. (BD, SafetyGlideTM, catalog number: 305918 )
    3. 3 ml plastic disposable transfer (Pasteur) pipettes, sterile in 20s (Elkay Laboratory Products, Liquipette®, catalog number: 127-P503-20S )
    4. 40 μm nylon cell strainer
    5. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
    6. 10 cm plate (100 x 20 mm) (Corning, Falcon®, catalog number: 353003 )
    7. 50 ml buffy coat from New York Blood Center (leukapharesis)
    8. Ficoll-Paque PLUS (GE Healthcare, HyCloneTM, catalog number: 17-1 440-02 )
    9. Dulbecco’s phosphate buffered saline (DPBS), without calcium, magnesium, phenol red (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
    10. Bovine serum albumin (BSA), fraction V, heat shock (Roche Diagnostics, catalog number: 03116956001 )
    11. EDTA
    12. RPMI 1640 media (GE Healthcare, HyCloneTM, catalog number: SH30096.01 )
    13. Fetal bovine serum (FBS)
    14. 1 M HEPES solution (GE Healthcare, HyCloneTM, catalog number: SH30237.01 )
    15. 2-mercaptoethanol (55 mM) (1,000x) (Thermo Fisher Scientific, GibcoTM, catalog number: 21985-023 )
    16. HyCloneTM L-glutamine (GE Healthcare, HyCloneTM, catalog number: SH3003401 )
    17. HyCloneTM penicillin streptomycin 100x solution (GE Healthcare, HyCloneTM, catalog number: SV30010 )
    18. Geneticin® selective antibiotic (G418 sulfate) (powder) (Thermo Fisher Scientific, GibcoTM, catalog number: 11811023)
    19. Anti-human CD14 microbeads
    20. 20. Human GM-CSF (Affymetrix, eBioscience, catalog number: 34-8339-82 )
    21. Human IL-4 (Affymetrix, eBioscience, catalog number: 34-8049-82 )
    22. Anti-hDC-SIGN PE (Clone: 120507), mouse IgG2B (R&D Systems, catalog number: FAB161P )
    23. Anti-hCD14 APC (Clone: 61D3) (Affymetrix, eBioscience, catalog number: 17-0149-42 )
    24. MACS buffer (see Recipes)
    25. DC medium (see Recipes)

  3. MDDC loading with HIV-1 or dextran
    1. 96 well clear round bottom TC-treated cell culture microplate, with lid, individually wrapped, sterile (Corning, Falcon®, catalog number: 353077 )
    2. X4-HIV-Gag-iGFP plasmid
      Note: Derived from HIV-1 molecular clone NL4-3 modified to insert the GFP protein in between the MA and CA domains of Gag as previously described (Hubner et al., 2007)
    3. Dextran, fluorescein (10,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1820 )
    4. Dextran, fluorescein (70,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1822 )
    5. Dextran, fluorescein (500,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-7136 )
    6. Dextran, fluorescein (2,000,000 MW, anionic, lysine fixable) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-7137 )
    7. Dulbecco’s phosphate buffered saline (DPBS), without calcium, magnesium, phenol red (GE Healthcare, HyCloneTM, catalog number: SH30028.02 )
    8. MACS buffer (see Recipes)

  4. Analysis of HIV-1 or dextran capture by flow cytometry
    1. Hanks’ balanced salt solution (HBSS) (1x) (Mediatech, catalog number: 21-023-CV )
    2. LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (for UV excitation) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L23105 )
    3. Anti-hDC-SIGN PE (Clone: 120507), mouse IgG2B (R&D Systems, catalog number: FAB161P )
    4. Anti-hCD14 APC (Clone: 61D3) (Affymetrix, eBioscience, catalog number: 17-0149-42 )
    5. Fixation/Permeabilization Solution Kit (RUO) (Fixation/Permeabilization solution 125 ml and BD Perm/WashTM buffer 100 ml) (BD, Cytofix/CytopermTM, catalog number: 554714 )
    6. Anti-P24 KC57-RD1 (Beckman Coulter, catalog number: 6604667 )

  5. Analysis of HIV-1 capture by Quantitative PCR (QPCR)
    1. LightCycler® 480 multiwell plate 96, white (Roche Diagnostics, catalog number: 04729692001 )
    2. LightCycler® 480 SYBR Green I Master (Roche Diagnostics, catalog number: 04887352001 )
    3. TRIzol® reagent (Thermo Fisher Scientific, AmbionTM, catalog number: 15596-018 )
    4. SuperScript® III first-strand synthesis system (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-051 )
    5. primers:
      GFP forward: 5’-ACGTAAACGGCCACAAGTTC-3’
      GFP Reverse: 5’-AAGTCGTGCTGCTTCATGTG-3’
      GAPDH forward: 5’-CCCATCACCATCTTCCAGGAGCG-3’
      GAPDH Reverse: 5’-GAGATGATGACCCTTTTGGCTCC-3’

  6. Analysis of HIV-1 or dextran capture by confocal microscopy
    1. FisherbrandTM microscope slides (25 x 75 x 1.0 mm, double frosted precleaned) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-552-5 )
    2. Cover slip, round, 5 mm diameter, #1 0211 glass (Corning, Falcon®, catalog number: CLS-1763-005 )
    3. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P8920-100ML )
    4. Paraformaldehyde, 16% solution, EM grade (Electron Microscopy Sciences, catalog number: 15710 )
    5. PIPES (1,4-piperazinediethanesulfonic acid) (Sigma-Aldrich, catalog number: P6757-100G )
    6. EGTA [Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid tetrasodium salt, ≥ 97%] (Sigma-Aldrich, catalog number: E8145-10G )
    7. Magnesium sulfate (MgSO4)
    8. Potassium hydroxide (KOH)
    9. TritonTM X-100 (Sigma-Aldrich, catalog number: X100-100ML )
    10. 5% casein solution as described and prepared in Dustin et al. (2007)
    11. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
    12. 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma-Aldrich, catalog number: D27802-25G )
    13. Poly(vinyl alcohol) (PVA) (Sigma-Aldrich, catalog number: 341584-25G )
    14. Glycerol (Sigma-Aldrich, catalog number: G5516-100ML )
    15. Tris buffer, pH 8.7 (1.5 M) (Bio-Rad Laboratories, catalog number: 1610798 )
    16. 2x PHEM buffer (see Recipes)
    17. DABCO-PVA (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5810 )
  2. Vortex
  3. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 240 )
  4. Flow cytometer (BD, model: LSRII )
  5. High throughput sampler (HTS) (BD)
  6. QPCR system (Roche Diagnostics, model: LightCycler 480 )
  7. Confocal microscope (Zeiss, model: LSM 710 )

Software

  1. ImageJ software

Procedure

  1. Production of HIV-1
    Note: HIV-1 particles are produced after calcium phosphate transfection in 293FT cells.
    1. 293FT cell culture
      1. 293FT cells are cultured in D10 medium in 10 cm plates, in the presence of 500 μg/ml of Geneticin®.
      2. When approaching confluence, cells are trypsinized (0.05% trypsin), washed by centrifugation (475 x g, 5 min at 4 °C) and plated after 1/5 dilution in 10 cm plates.
      3. Plate 8 x 106/15 cm plate, 293FT cells the day before transfection in 30 ml of D10 medium without Geneticin®.
      4. One 15 cm plate is going to produce 20 ml of virus, so transfection experiment should be scaled accordingly.
      Note: In order to maximize viral particle production, 293FT cells are never kept more than 3 weeks in culture (After 3 weeks, always go back to frozen starting aliquots).
    2. Calcium phosphate transfection
      Note: The Promega kit is used for calcium phosphate transfection. Transfection protocol is described for transfection in one 15 cm plate.
      1. Dilute 60 μg of DNA (X4-HIV-Gag-iGFP) with 1.12 ml nuclease-free H2O in a 15 ml conical tube (tube 1).
      2. Add 168 μl of CaCl2, 2 M, to the diluted DNA.
      3. In another tube (tube 2), add 1,350 μl of 2x HEPES-buffered saline (2x HBS).
      4. While gently vortexing tube 2, add dropwise the prepared DNA solution to the HBS in tube 2.
      5. Incubate the solution at room temperature for 30 min.
      6. Vortex the transfection solution again just before adding it to the cells.
      7. Add the solution containing DNA- CaCl2-HBS, dropwise in various places around the plate.
      8. Swirl the plate to distribute the solution evenly.
      9. 20 h after transfection, carefully replace the culture medium with 20 ml of D10 medium without G418.
      10. 24 h after medium change, harvest the supernatant.
      11. Filter the supernatant twice (to remove cells and debris) with 0.45 μm sterile filter unit with PVDF membrane.
      12. Filtered viral supernatant can be used fresh or stored at -80 °C.

  2. Differentiation of monocyte derived dendritic cells (MDDCs)
    1. Obtaining peripheral blood mononuclear cells (PBMCs). PBMCs are obtained from buffy coats ordered from the New York Blood Center using mononuclear cell isolation with Ficoll-Paque separation media.
    2. Ficoll and DPBS are brought to room temperature (RT).
    3. Recover blood from buffy coat using 60 ml syringe and 18 G thick needle.
    4. Mix blood with RT DPBS (1:1 ratio).
    5. Add 25 ml of diluted blood using a needle to a 50 ml Falcon tube already containing 15 ml of Ficoll.
    6. Centrifuge for 30 min at 845 x g, RT and with brake off.
    7. Recover the white leukocyte layer (in between the yellow serum and the red platelets layers) with a plastic pipette.
    8. Wash the leukocyte layers containing the PBMCs with cold DPBS, centrifuge at 170 x g for 10 min at 4 °C (slow spin to remove platelets).
    9. Resuspend the cells in MACS buffer, wash, spin at 475 x g, 5 min at 4 °C.
    10. Count the cells.
    11. Add 50 μl of anti-human CD14 microbeads from Miltenyi and 950 μl MACS buffer per 100 million PBMCs.
    12. 20 min incubation at 4 °C.

    Note: While doing the staining of PBMCs with anti CD14 magnetic beads, always do the staining in the fridge rather than on ice in order to prevent antibody ‘capping effect’.

    1. Wash in MACS buffer, then spin at 475 x g, 5 min at 4 °C.
    2. Resuspend in 5 ml MACS buffer and filter using a 40 μm nylon cell strainer.
    3. Run the sample through an LS MACS separation column for positive selection.
    4. Perform 3 washes of the LS column, using 3 ml of MACS buffer.
    5. Recover the CD14+ cells from the column by using 5 ml of MACS buffer and a plunger.
    6. Count the cells and plate them at a concentration of 1 million/ml in DC medium with hGM-CSF (10 ng/ml) and hIL-4 (50 ng/ml).
      Note: To obtain optimal results regarding monocyte differentiation into MDDCs, plate 20 million monocytes per 10 cm plate, in 20 ml DC medium.
    7. 2 days post isolation, add 40% of new DC medium with hGM-CSF (10 ng/ml) and hIL-4 (50 ng/ml).
    8. 4 days post isolation, check MDDC differentiation by flow cytometry: monitor upregulation of DC-SIGN and downregulation of CD14 (1/200 of stock solution for both antibodies). 

  3. MDDC loading with HIV-1 or dextran
    1. Plate 50,000 MDDCs (1 million/ml) per well in a U bottom 96-well plate.
    2. Place the 96-well plate on ice, while adding HIV-1 or dextran molecules, for synchronization of capture.
    3. Add 50 ng of HIV-1 or 100 μg/ml of FITC-labeled dextran molecules of various size (10kDa, 70kDa, 500kDa, 2,000kDa).
      Note: Don’t forget to add negative controls: no virus or dextran and/or cells staying at 4 °C.
    4. Incubate at 37 °C, 5% CO2, for 4 and 24 h for HIV-1 and 30 min and 4 h for dextran molecules.
    5. After incubation, wash extensively by adding 200 μl cold DPBS and centrifuge 5 min, 475 x g at 4 °C.
    6. Repeat the washing step 3 times.
    7. Analyze HIV-1 or dextran capture.

  4. Analysis of HIV-1 or dextran capture by flow cytometry
    1. For flow cytometry analysis, take the cells loaded with dextran or HIV-1.
    2. Stain with 50 μl of a live/dead blue fixable dye diluted in HBSS (1x) (1/500) 15 min on ice.
    3. Wash using MACS buffer, 475 x g, 5 min, 4 °C.
    4. Stain with anti-hDC-SIGN-PE (1/200 diluted in MACS buffer) and anti-hCD14-APC (1/200 diluted in MACS buffer) to control for MDDC differentiation.
    5. Incubate for 30 min, 4 °C, covered.
    6. Wash using MACS buffer, 475 x g, 5 min, 4 °C.
    7. Resuspend in 100 μl.
    8. For dextran detection, no further staining is required. For HIV-1 detection, cells need to be fixed, using 50 μl of BD Cytofix/CytopermTM, 20 min at 4 °C.
    9. Wash using 100 μl of BD perm/Wash buffer.
    10. Stain for HIV-1 capsid using anti-P24-RD1, diluted 1/500 in BD perm/Wash buffer. 1 h at RT.
    11. Wash using MACS buffer, 475 x g, 5 min, 4 °C.
    12. Resuspend in 100 μl.
    13. Proceed to flow cytometry analysis.
      Note: For flow cytometry, BD high throughput sampler (HTS) can be used to run 96-well plates.

  5. Analysis of HIV-1 capture by Quantitative PCR (QPCR)
    Real time quantitative PCR (qPCR) was performed after reverse transcription using a Roche LightCycler 480 with Roche 480 SYBR Green I Master reagent according to manufacturer specifications. The relative abundance of HIV is calculated based on the amount of GFP mRNA (the virus encodes GFP) using a standard curve and normalized using GAPDH as a control.
    1. Total RNA is extracted from 3 x 50,000 MDDCs using TRIZOL® RNA isolation protocol.
    2. 5 μl of RNA is reverse transcribed using SuperScript® III first-strand synthesis system in a 20 μl final reaction volume.
    3. For each sample, 2 μl of diluted cDNA (dilution in water, 1/2 and 1/5) is used for QPCR reaction.
    4. QPCR mix:
      6 μl of 2x SYBR Green mix
      0.6 μl primer F (10 μM)
      0.6 μl primer R (10 μM)
      2.8 μl of H2O
      2 μl of cDNA
    5. QPCR reaction:
      Step 1: 95 °C, 5 min
      Step 2: 95 °C, 5 sec
      Step 3: 60 °C, 30 sec
      Step 4: 72 °C, 20 sec
      Back to step 2, 50 cycles
    6. QPCR analysis: based on standard curve, determine GFP mRNA quantity and normalize it by GAPDH in each sample.

  6. Analysis of HIV-1 or dextran capture by confocal microscopy (Figures 1 and 2)
    1. MDDCs loaded with HIV-1 or dextran are put on coverslips previously coated with poly-L-lysine solution, for 30 min at 37 °C.
    2. Phalloidin staining (to detect cortical actin), or any cell surface staining, is performed in PBS-1% BSA for 30 min at room temperature (Figure 2).
    3. For fixation, incubate with warm paraformaldehyde A (PFA) (2%) in PHEM buffer, for 10 min at room temperature.
    4. Permeabilize in 0.1% Triton X-100 diluted in PHEM buffer, for 5 min at RT.
    5. Blocking is done in 5% casein solution, 1 h at 4 °C.
    6. For intracellular staining (if needed), antibodies are diluted in 5% casein solution and incubated for 1 h at room temperature.
    7. For nuclear detection, cells are then stained with DAPI, (1/5,000 dilution from stock at 5 mg/ml in H2O), at RT (Figures 1 and 2).
    8. Upon 5 washing (200 μl each) in PBS-0.1% BSA, cells are mounted in a homemade DABCO-PVA medium.
    9. Using a confocal microscope, analyze for each cell the intensity of internalized GFP or FITC, which represent, respectively, captured HIV-1 particles and dextran molecules. To do so, acquire 400 nm Z-stacks and determine the HIV-1 and dextran molecules. Quantification of HIV-1 and dextran uptake will be discussed in the Data analysis section. (Figures 1 and 2)


      Figure 1. Example of untransduced and untreated MDDCs loaded for 4 h with 100 μg/ml of 10 kDa FITC-labeled dextran molecules. Cells on coverslips can be seen on the left panel (bright-field). On the right panel, one Z-stack section of 400 nm is displayed where nuclei of cells were stained with DAPI (blue) and molecules of dextran can be visualized in green.


      Figure 2. Example of HIV-1 internalization by confocal microscopy. MDDCs untransduced (top panel) or transduced with a shRNA against DNM2 (bottom panel) loaded for 4 h with 50 ng of HIV-1 (green dots). Bright-field can be seen on the left panel. On the right panel, one Z-stack section of 400 nm is displayed where nuclei of cells were stained with DAPI (blue), actin filaments in red, after phalloidin staining and captured HIV-1 in green.

Data analysis

  1. Analysis of HIV-1 or dextran capture by flow cytometry (Figure 3)
    1. First, the gate must be set on live cells, by excluding cells that have incorporated the live/dead stain. It is important that MDDCs are well differentiated, as assessed by high expression of DC-SIGN and low expression of CD14. (Figure 3A)
    2. Monocytes are usually CD14 high and DC-SIGN low.
    3. The amount of HIV-1 or dextran internalized can be calculated by comparing the mean fluorescence intensity (MFI) to the negative control and determining the ratio of internalization between samples (Figure 3B).
    4. It is recommended that HIV and dextran capture be assessed by using different dilutions and different times of incubation, in order to draw a representative and more accurate curve.
    5. Each sample is usually analyzed in triplicate and the experiment is performed with cells coming from at least 3 independent healthy blood donors.
    6. Statistical analyses of the differences in MFI between samples can be done using the Holm-Sidak multiple comparison test following one-way ANOVA (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).


      Figure 3. Analysis of HIV-1 and dextran capture by flow cytometry. A. Gating strategy to analyse MDDCs: left panel, gating on MDDCs based on size (FSC) and granularity (SSC); right panel gating on alive differentiated MDDCs (DC-SIGN+; dapi-). B. Example of histograms generated after gating on MDDCs loaded with HIV-1 (top panel) or dextran (lower panel). In grey, an isotype control for P24 is used to assess the amount of HIV-1 captured by MDDCs transduced with scramble shRNA vs. TSPAN7 shRNA. For dextran internalization, the grey histograms represent a negative control where cells are staying at 4 °C to prevent internalization.

  2. Analysis of HIV-1 capture by Quantitative PCR (QPCR) (Figure 4)
    1. After reverse transcription, the amount of HIV-1 RNA was detected by quantitative PCR with primers for GFP (encoded inside the HIV genome) and normalized to GAPDH.
    2. To assess the quantity in the most accurate way possible, every experiment should include a standard curve using the GFP primers and also the GAPDH primers.
    3. Then the standard curve is used to determine the quantity of HIV internalized in each sample and compare that to the negative controls (no virus or capture experiment performed at 4 °C).
    4. Normalized HIV-1 quantity can be compared between different samples to assess the increase or the decrease in HIV capture (Figure 4).
    5. Due to experimental variations that could exist between different blood donors, it is preferable not to compare samples coming from different donors, but seek consistency between donors in terms of trends. Experiments should be done in triplicate and in at least five different blood donors.
    6. Statistical analyses of the differences in HIV capture between samples can be done using the Holm-Sidak multiple comparison test following one-way ANOVA (NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) (Figure 4).


      Figure 4. Analysis of HIV-1 capture by quantitative PCR. Example of histograms after HIV capture experiments and analysis by quantitative PCR. MDDCs transduced with scramble shRNA, empty vectors or shRNA against TSPAN7 were loaded with HIV-GFP for 4 or 24 h. Quantity of HIV-1 captured is analysed by PCR against GFP, and compared to the quantity captured after scramble shRNA (set arbitrarily to a value of 1).

  3. Analysis of HIV-1 or dextran capture by confocal microscopy (Figure 5)
    1. For quantification of dextran internalization by confocal microscopy, we use phalloidin staining (for filamentous actin) to identify the outside borders of every cell.
      1. Using the ImageJ software, the maximal intensity of dextran per pixel is collected for each Z-stack.
      2. The total intensity of dextran is then obtained and normalized to the volume analyzed.
      3. The integrated intensity can be calculated by taking the sum of all voxels, at each intensity greater than background (intensity ≥ 200), then multiply by the intensity and summing.
      4. Volume is obtained by counting all voxels greater than background.
      5. Concentration is the integrated intensity divided by volume (arbitrary units) (Figure 5).
      6. For each sample, we recommend to calculate the concentration of dextran for at least 100 cells and to repeat the experiment with at least 3 different human blood donors. Unpaired t-test can be used to determine statistical significance.
    2. Regarding quantification of HIV-1 internalization, 400 nm Z-stacks are acquired using a confocal microscope with an average of 20 Z-stacks per cell.
      1. Every HIV-1 particle (green dot) can be manually counted as internalized based on its localization compared to the cortical actin barrier (phalloidin staining).
      2. Based on electron microscopy data, an average of 5 HIV viral particles was estimated to be present per HIV-1 aggregate.
      3. For each cell, the total of HIV-1 can thus be calculated and an average of at least 100 cells per sample is analyzed. Unpaired t-test can be used to determine statistical significance.


        Figure 5. Analysis of dextran capture by confocal microscopy. MDDCs transduced with a scramble shRNA or shRNA against TSPAN7 were cultured for 4 h with 10 kDa (left panel) or 70 kDa molecules of dextran (right panel). The concentration of dextran internalized by each MDDC was then determined as explained above. Briefly, concentration is the integrated density of dextran staining divided by volume and is displayed with arbitrary units on the y axis. For each condition, each dot represents a different cell and the average is displayed ± SEM.

Notes

  1. In general, when working with human primary cells, you need to make sure to have internal controls in order to be able to normalize the data and to compare data coming from different blood donors. Variation can also be observed from donor to donor and increasing the number of different human blood donors usually helps to have better analysis of the data.
  2. We recommend to always check the status of maturation of MDDCs. Experiments as described in this protocol are performed using immature MDDCs. Once mature, the results obtained in terms of HIV-1 capture and dextran internalization are different. Maturation status can be determined by looking at CD86 and CD80 molecules by flow cytometry. Immature MDDCs should be CD80 and CD86 low.
  3. All 3 techniques measure HIV-1 internalization by MDDCs. Combining 3 different methods is increasing the chances to detect even small differences in capture and internalization and is increasing the robustness of the results. To detect changes in HIV-1 internalization, flow cytometry is maybe not the best method as it doesn’t seem to be as sensitive as QPCR or confocal microscopy to detect small amount of HIV internalization. Quantitative PCR is maybe the fastest and easiest method but is not allowing a direct comparison of HIV-1 and dextran internalization and any artefactual contamination could potentially biased the data. The most demanding technique is probably the confocal microscopy but allows the most rigorous comparison of both HIV-1 and dextran internalization between different samples.

Recipes

  1. D10 medium
    DMEM
    10% fetal bovine serum (FBS, heat inactivated)
    0.1 mM MEM non-essential amino acids
    6 mM L-glutamine
    1 mM MEM sodium pyruvate
    100 U/ml penicillin
    100 μg/ml streptomycin
    50 μg/ml gentamycin
  2. MACS buffer
    DPBS
    0.5% bovine serum albumin (BSA)
    1 mM EDTA
  3. DC medium
    RPMI
    10% FBS (heat inactivated)
    10 mM Hepes (Hyclone)
    55 μM β-mercaptoethanol
    6 mM L-glutamine
    100 U/ml penicillin
    100 μg/ml streptomycin
    50 μg/ml gentamycin
  4. 2x PHEM buffer (500 mls)
    18.14 g PIPES
    6.5 g HEPES
    3.8 g EGTA
    0.99 g MgSO4
    Adjust pH to 7.0 with 10 N KOH
  5. DABCO-PVA
    2.5% Dabco
    10% polyvinylalcohol (PVA)
    5% glycerol
    25 mM Tris buffer, pH 8.7

Acknowledgments

We thank Alice F. Liang, Michael Cammer and the NYULMC OCS Microscopy Core for the service provided for light microscopy; Wendy Lin for technical help; and Jarrod Johnson, Nicolas Manel, for their critical advice. This work was supported by fellowships from EMBO, the Cancer Research Institute, and the Philippe Foundation (M.M.M.); by the Howard Hughes Medical Institute (D.R.L.) and Helen and Martin Kimmel Center for Biology and Medicine (D.R.L.); and by grants from the National Institutes of Health (R21AI084633) (D.R.L.) and NCRR (S10RR023704-01A1).

References

  1. Dustin, M. L., Starr, T., Varma, R. and Thomas, V. K. (2007). Supported planar bilayers for study of the immunological synapse. Curr Protoc Immunol Chapter 18: Unit 18 13.
  2. Hubner, W., Chen, P., Del Portillo, A., Liu, Y., Gordon, R. E. and Chen, B. K. (2007). Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1. J Virol 81(22): 12596-12607.
  3. Menager, M. M. and Littman, D. R. (2016). Actin dynamics regulates dendritic cell-mediated transfer of HIV-1 to T cells. Cell 164(4): 695-709.

简介

本协议的目的是描述如何使用三种不同的技术:流式细胞术,定量PCR和共聚焦显微镜来测量和量化人类单核细胞衍生树突细胞(MDDCs)中内在的HIV-1颗粒和葡聚糖分子的量。

[背景] 开发此协议是为了评估在人单核细胞衍生的树突细胞中肌动蛋白成核破坏时HIV-1内化的变化。在shRNA筛选后,识别对于HIV-1从树突状细胞转移到T细胞重要的基因,我们观察到肌动蛋白成核的中断导致从富含肌动蛋白的树突到水泡的转变,由于过量的肌动球蛋白收缩。结果,观察到HIV-1转移的减少和由于水泡收缩驱动的巨噬细胞增多引起的HIV-1内化的增加。我们的结论是,肌动蛋白成核和稳定的效应器是维持艾滋病毒1对肌动蛋白丰富的树突和限制其内吞作用,有效转移到T淋巴细胞的关键(Menager和Littman,2016)。

材料和试剂

  1. HIV-1颗粒的生产
    1. 10cm板(100×20mm)(Corning,Falcon ,目录号:353003)
    2. 15cm板(150×20mm)(Corning,Falcon ,目录号:353025)
    3. 15 ml锥形管
    4. 33mm Millex-HV注射器过滤器,PVDF,0.45μm(EMD Millipore,目录号:SLHV033RS)
    5. 具有BD Luer-Lok 尖端(BD,Luer-Lok TM ,目录号:309653)的60ml注射器
    6. 50ml高清晰度PP离心管,锥形底,无菌,25 /袋,500 /箱(Corning,Falcon ,目录号:352070)
    7. 293FT细胞(Thermo Fisher Scientific,Invitrogen TM,目录号:R700-07)
    8. X4-HIV-Gag-iGFP质粒 注意:衍生自HIV-1分子克隆NL4-3,其经修饰以如先前所述在Gag的MA和CA结构域之间插入GFP蛋白(Hubner等人,2007)。
    9. Dulbecco对Eagles培养基(DMEM)的修饰(Mediatech,目录号:10-017-CV)
    10. 胎牛血清(FBS)
    11. MEM氨基酸溶液(GE Healthcare,HyClone ,目录号:SH30598.01)
    12. HyClone TM L-谷氨酰胺(GE Healthcare,HyClone ,目录号:SH3003401)
    13. HyClone 青霉素链霉素100x溶液(GE Healthcare,HyClone ,目录号:SV30010)
    14. 庆大霉素(50mg/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15750060)
    15. 遗传霉素选择性抗生素(G418硫酸盐)(粉末)(Thermo Fisher Scientific,Gibco TM ,目录号:11811023)
    16. 胰蛋白酶0.05%蛋白酶溶液与猪胰蛋白酶,HBSS,EDTA;无钙,镁(GE Healthcare,HyClone ,目录号:SH30236.01)。
    17. ProFection 哺乳动物转染系统 - 磷酸钙(Promega,目录号:E1200)
    18. 氯化钙(CaCl 2)
    19. 100mM丙酮酸钠溶液(GE Healthcare,HyClone ,目录号:SH30239.01)
    20. 1 M HEPES溶液(GE Healthcare,HyClone ,目录号:SH30237.01)
    21. D10介质(见配方)

  2. 单核细胞衍生的树突细胞(MDDC)的分化
    1. 具有BD Luer-Lok 尖端(BD,Luer-Lok TM ,目录号:309653)的60ml注射器
    2. 针(50/sp,500/ca),18G×1/2英寸(BD,SafetyGlide TM ,目录号:305918)
    3. 3ml塑料一次性转移(Pasteur)移液管,20s无菌(Elkay Laboratory Products,Liquipette ,目录号:127-P503-20S)
    4. 40μm尼龙细胞过滤器
    5. LS柱(Miltenyi Biotec,目录号:130-042-401)
    6. 10cm板(100×20mm)(Corning,Falcon ,目录号:353003)
    7. 50毫升来自纽约血液中心的白细胞层(白血病)
    8. Ficoll-Paque PLUS(GE Healthcare,HyClone ,目录号:17-1 440-02)
    9. 不含钙,镁,酚红的Dulbecco's磷酸盐缓冲盐水(DPBS)(GE Healthcare,HyClone ,目录号:SH30028.02)
    10. 牛血清白蛋白(BSA),级分V,热休克(Roche Diagnostics,目录号:03116956001)
    11. EDTA
    12. RPMI 1640培养基(GE Healthcare,HyClone ,目录号:SH30096.01)
    13. 胎牛血清(FBS)
    14. 1 M HEPES溶液(GE Healthcare,HyClone ,目录号:SH30237.01)
    15. 2-巯基乙醇(55mM)(1,000x)(Thermo Fisher Scientific,Gibco TM,目录号:21985-023)
    16. HyClone TM L-谷氨酰胺(GE Healthcare,HyClone ,目录号:SH3003401)
    17. HyClone 青霉素链霉素100x溶液(GE Healthcare,HyClone ,目录号:SV30010)
    18. 遗传霉素选择性抗生素(G418硫酸盐)(粉末)(Thermo Fisher Scientific,Gibco TM ,目录号:11811023)
    19. 抗人CD14微珠
    20. 20.人GM-CSF(Affymetrix,eBioscience,目录号:34-8339-82)
    21. 人IL-4(Affymetrix,eBioscience,目录号:34-8049-82)
    22. 抗hDC-SIGN PE(克隆:120507),小鼠IgG2B(R& D Systems,目录号:FAB161P)
    23. 抗hCD14 APC(克隆:61D3)(Affymetrix,eBioscience,目录号:17-0149-42)
    24. MACS缓冲区(参见配方)
    25. DC介质(参见配方)

  3. MDDC加载HIV-1或葡聚糖
    1. 96孔透明的圆底TC-处理的细胞培养微板,带有盖,单独包装,无菌(Corning,目录号:353077)
    2. X4-HIV-Gag-iGFP质粒 注意:衍生自HIV-1分子克隆NL4-3,如先前所述(Hubner等人,2007)修饰以将GFP蛋白插入Gag的MA和CA结构域之间
    3. 葡聚糖,荧光素(10,000MW,阴离子,赖氨酸可固定)(Thermo Fisher Scientific,Molecular Probes ,目录号:D-1820)
    4. 葡聚糖,荧光素(70,000MW,阴离子,赖氨酸可固定)(Thermo Fisher Scientific,Molecular Probes ,目录号:D-1822)
    5. 葡聚糖,荧光素(500,000MW,阴离子,赖氨酸可固定)(Thermo Fisher Scientific,Molecular Probes ,目录号:D-7136)
    6. 葡聚糖,荧光素(2,000,000MW,阴离子,赖氨酸可固定)(Thermo Fisher Scientific,Molecular Probes ,目录号:D-7137)
    7. 不含钙,镁,酚红的Dulbecco's磷酸盐缓冲盐水(DPBS)(GE Healthcare,HyClone ,目录号:SH30028.02)
    8. MACS缓冲区(参见配方)

  4. 通过流式细胞术分析HIV-1或葡聚糖捕获
    1. Hanks平衡盐溶液(HBSS)(1x)(Mediatech,目录号:21-023-CV)
    2. LIVE/DEAD 可固定的蓝色死细胞染色试剂盒(用于UV激发)(Thermo Fisher Scientific,Molecular Probes TM ,目录号:L23105)
    3. 抗hDC-SIGN PE(克隆:120507),小鼠IgG2B(R& D Systems,目录号:FAB161P)
    4. 抗hCD14 APC(克隆:61D3)(Affymetrix,eBioscience,目录号:17-0149-42)
    5. 固定/渗透溶液试剂盒(RUO)(固定/渗透溶液125ml和BD Perm/Wash TM缓冲液100ml)(BD,Cytofix/Cytoperm TM,目录号: 554714)
    6. 抗-P24 KC57-RD1(Beckman Coulter,目录号:6604667)

  5. 通过定量PCR(QPCR)对HIV-1捕获的分析
    1. 白色(Roche Diagnostics,目录号:04729692001)的LightCycler 480多孔板96.
    2. LightCycler 480 SYBR Green I Master(Roche Diagnostics,目录号:04887352001)
    3. TRIzol试剂(Thermo Fisher Scientific,Ambion TM ,目录号:15596-018)
    4. SuperScript III第一链合成系统(Thermo Fisher Scientific,Invitrogen TM ,目录号:18080-051)
    5. 引物:
      GFP正向:5'-ACGTAAACGGCCACAAGTTC-3'
      GFP反向:5'-AAGTCGTGCTGCTTCATGTG-3'
      GAPDH正向:5'-CCCATCACCATCTTCCAGGAGCG-3'
      GAPDH反向:5'-GAGATGATGACCCTTTTGGCTCC-3'

  6. 通过共聚焦显微镜分析HIV-1或葡聚糖捕获
    1. (Thermo Fisher Scientific,Fisher Scientific,目录号:12-552-5)的Fisherbrand TM显微镜载玻片(25×75×1.0mm,双磨砂预清洗)
    2. 盖玻片,圆形,5mm直径,#1 0211玻璃(Corning,Falcon ,目录号:CLS-1763-005)
    3. 聚-L-赖氨酸溶液(Sigma-Aldrich,目录号:P8920-100ML)
    4. 多聚甲醛,16%溶液,EM级(Electron Microscopy Sciences,目录号:15710)
    5. PIPES(1,4-哌嗪二乙磺酸)(Sigma-Aldrich,目录号:P6757-100G)
    6. EGTA [乙二醇 - 双(β-氨基乙基醚)-N,N,N',N'-四乙酸四钠盐,≥97%](Sigma-Aldrich,目录号:E8145-10G)
    7. 硫酸镁(MgSO 4)
    8. 氢氧化钾(KOH)
    9. TritonX-100(Sigma-Aldrich,目录号:X100-100ML)
    10. 5%酪蛋白溶液,如在Dustin等人中描述和制备的。 (2007)
    11. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Thermo Fisher Scientific,Molecular Probes ,目录号:D1306)
    12. 1,4-二氮杂双环[2.2.2]辛烷(DABCO)(Sigma-Aldrich,目录号:D27802-25G)
    13. 聚(乙烯醇)(PVA)(Sigma-Aldrich,目录号:341584-25G)
    14. 甘油(Sigma-Aldrich,目录号:G5516-100ML)
    15. Tris缓冲液,pH8.7(1.5M)(Bio-Rad Laboratories,目录号:1610798)
    16. 2x PHEM缓冲液(参见配方)
    17. DABCO-PVA(参见配方)

设备

  1. 离心机(Eppendorf,型号:5810)
  2. 涡流
  3. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Heracell TM 240)
  4. 流式细胞仪(BD,型号:LSRII),这链接到HTS模块,而不是LSRII
  5. 高通量取样器(HTS)(BD)
  6. QPCR系统(Roche Diagnostics,型号:LightCycler 480)
  7. 共焦显微镜(Zeiss,型号:LSM 710)

软件

  1. ImageJ软件

程序

  1. HIV-1的生产
    注意:HIV-1颗粒是在293FT细胞中磷酸钙转染后产生的。
    1. 293FT细胞培养
      1. 在500μg/ml的Geneticin存在下,将293FT细胞在10cm平板中的D10培养基中培养。
      2. 当接近汇合时,将细胞胰蛋白酶化(0.05%胰蛋白酶),通过离心(475×g,在4℃下5分钟)洗涤,并在1/5稀释后铺板在10cm平板中。
      3. 板8×10 6/15/15cm平板,293FT细胞在转染前一天在30ml不含Geneticin的D10培养基中。
      4. 一个15厘米板将产生20毫升病毒,所以转染实验应相应地缩放。
      注意:为了最大化病毒颗粒的产生,293FT细胞在培养过程中永远不会超过3周(3周后,总是回到冷冻的起始等分试样)。
    2. 磷酸钙转染
      注意:Promega试剂盒用于磷酸钙转染。描述了用于在一个15cm平板中转染的转染方案。
      1. 在15ml锥形管(试管1)中用1.12ml无核酸酶的H 2 O 2稀释60μgDNA(X4-HIV-Gag-iGFP)。
      2. 向稀释的DNA中加入168μlCaCl 2,2M,
      3. 在另一个管(管2)中,加入1350μl的2x HEPES缓冲盐水(2x HBS)
      4. 在轻轻涡旋管2的同时,将制备的DNA溶液滴加到管2中的HBS
      5. 将溶液在室温下孵育30分钟
      6. 在将转染溶液加入细胞之前再次涡旋转染溶液
      7. 在板周围的各个位置滴加含有DNA-CaCl 2 HBS的溶液。
      8. 旋转板以均匀地分布溶液。
      9. 转染后20小时,用20ml不含G418的D10培养基小心地更换培养基
      10. 培养基更换后24小时,收获上清液
      11. 用0.45μm无菌过滤单元用PVDF膜过滤上清液两次(以除去细胞和碎片)
      12. 过滤的病毒上清液可以新鲜使用或储存于-80℃
  2. 单核细胞衍生的树突细胞(MDDC)的分化
    1. 获得外周血单核细胞(PBMC)。使用从纽约血液中心订购的血沉棕黄层,使用Ficoll-Paque分离介质的单核细胞分离获得PBMC。
    2. 使Ficoll和DPBS达到室温(RT)
    3. 使用60毫升注射器和18 G厚的针从血沉棕黄层中恢复血液
    4. 将血液与RT DPBS(1:1比例)混合
    5. 使用针头加入25毫升稀释的血液到已经含有15毫升Ficoll的50毫升Falcon管
    6. 在845×g离心30分钟,RT,并且制动关闭。
    7. 用塑料移液管回收白色白细胞层(黄色血清和红色血小板层之间)。
    8. 用冷的DPBS洗涤含有PBMC的白细胞层,在4℃下以170×g离心10分钟(缓慢旋转以除去血小板)。
    9. 将细胞重悬在MACS缓冲液中,洗涤,在475℃下旋转,在4℃下5分钟。
    10. 计数单元格。
    11. 每100万个PBMC加入50μl来自Miltenyi的抗人CD14微珠和950μlMACS缓冲液。
    12. 在4℃孵育20分钟。

    注意:在用抗CD14磁珠染色PBMC时,总是在冰箱中而不是在冰上染色,以防止抗体的上限效应。

    1. 在MACS缓冲液中洗涤,然后在475℃下旋转,在4℃下5分钟。
    2. 重悬于5ml MACS缓冲液中,并使用40μm尼龙细胞滤器过滤
    3. 通过LS MACS分离柱运行样品进行阳性选择
    4. 使用3ml MACS缓冲液进行LS柱的3次洗涤
    5. 通过使用5毫升MACS缓冲液和柱塞从柱中恢复CD14 + 细胞。
    6. 计数细胞,并在具有hGM-CSF(10ng/ml)和hIL-4(50ng/ml)的DC培养基中以1百万/ml的浓度平板。
      注意:为了获得关于单核细胞分化成MDDC的最佳结果,在20mL DC培养基中,每10cm平板接种2000万单核细胞。
    7. 分离后2天,加入40%的具有hGM-CSF(10ng/ml)和hIL-4(50ng/ml)的新DC培养基。
    8. 分离后4天,通过流式细胞术检查MDDC分化:监测DC-SIGN的上调和CD14的下调(两种抗体的1/200的储备溶液)。 

  3. MDDC加载HIV-1或葡聚糖
    1. 在U底96孔板中每孔加入50,000个MDDC(1百万/ml)。
    2. 将96孔板放在冰上,同时加入艾滋病毒1或葡聚糖分子,同步捕获
    3. 加入50ng的HIV-1或100μg/ml的各种大小的FITC标记的葡聚糖分子(10kDa,70kDa,500kDa,2,000kDa)。
      注意:不要忘记添加阴性对照:没有病毒或葡聚糖和/或细胞保持在4°C。
    4. 在37℃,5%CO 2孵育4和24小时HIV-1和30分钟和4小时葡聚糖分子。
    5. 温育后,通过加入200μl冷DPBS广泛洗涤,并在4℃离心5分钟,475×g。
    6. 重复洗涤步骤3次。
    7. 分析HIV-1或葡聚糖捕获
  4. 通过流式细胞术分析HIV-1或葡聚糖捕获
    1. 对于流式细胞术分析,取带有葡聚糖或HIV-1的细胞
    2. 用50μl在HBSS(1x)(1/500)中稀释的活/死蓝可固定染料在冰上染色15分钟。
    3. 使用MACS缓冲液(475×g)洗涤,5分钟,4℃
    4. 用抗hDC-SIGN-PE(在MACS缓冲液中稀释1/200)和抗hCD14-APC(在MACS缓冲液中稀释1/200)染色以控制MDDC分化。
    5. 孵育30分钟,4℃,盖上
    6. 使用MACS缓冲液(475×g)洗涤,5分钟,4℃
    7. 重悬于100μl。
    8. 对于葡聚糖检测,不需要进一步染色。对于HIV-1检测,需要使用50μlBD Cytofix/Cytoperm TM ,在4℃下20分钟固定细胞。
    9. 使用100μl的BD Perm/Wash缓冲液洗涤
    10. 使用抗P24-RD1,在BD Perm/Wash缓冲液中1/500稀释的HIV-1衣壳的染色。室温下1小时。
    11. 使用MACS缓冲液(475×g)洗涤,5分钟,4℃
    12. 重悬于100μl。
    13. 继续流式细胞术分析。
      注意:对于流式细胞术,可使用BD高通量取样器(HTS)运行96孔板。

  5. 通过定量PCR(QPCR)对HIV-1捕获的分析
    使用Roche LightCycler 480和Roche 480 SYBR Green I Master试剂根据制造商说明书在逆转录后进行实时定量PCR(qPCR)。使用标准曲线,基于GFP mRNA(病毒编码GFP)的量计算HIV的相对丰度,并使用GAPDH作为对照进行标准化。</i>
    1. 使用TRIZOL RNA分离方案从3×50,000个MDDC中提取总RNA。
    2. 使用SuperScript III第一链合成系统在20μl最终反应体积中逆转录5μlRNA。
    3. 对于每个样品,将2μl稀释的cDNA(在水中稀释,1/2和1/5)用于QPCR反应
    4. QPCR混合:
      6μl2x SYBR Green混合物
      0.6μl引物F(10μM)
      0.6μl引物R(10μM)
      2.8μlH 2 O x/b 2μlcDNA
    5. QPCR反应:
      步骤1:95℃,5分钟
      步骤2:95℃,5秒
      步骤3:60℃,30秒
      步骤4:72℃,20秒
      返回步骤2,50个周期
    6. QPCR分析:基于标准曲线,确定GFP mRNA量并通过GAPDH在每个样品中归一化
  6. 通过共聚焦显微镜分析HIV-1或葡聚糖捕获(图1和图2)
    1. 将装有HIV-1或葡聚糖的MDDC放在预先用聚-L-赖氨酸溶液包被的盖玻片上,在37℃下孵育30分钟。
    2. 在室温下在PBS-1%BSA中进行鬼笔环肽染色(以检测皮层肌动蛋白)或任何细胞表面染色30分钟(图2)。
    3. 对于固定,在PHEM缓冲液中温热的多聚甲醛A(PFA)(2%)在室温下孵育10分钟。
    4. 在0.1%Triton X-100中在PHEM缓冲液中稀释5分钟,在室温下渗透
    5. 在5%酪蛋白溶液中进行封闭,在4℃下进行1小时
    6. 对于细胞内染色(如果需要),将抗体在5%酪蛋白溶液中稀释并在室温下温育1小时。
    7. 对于核检测,然后在室温下用DAPI(在H 2 O中以5mg/ml的储备液稀释1/5,000稀释)将细胞染色。(图1和2)。
    8. 在PBS-0.1%BSA中洗涤5次(每次200μl)后,将细胞安装在自制的DABCO-PVA培养基中。
    9. 使用共聚焦显微镜,为每个细胞分析内化GFP或FITC的强度,其分别代表捕获的HIV-1颗粒和葡聚糖分子。为此,获得400 nm Z堆叠,并确定艾滋病毒1和葡聚糖分子。 HIV-1和葡聚糖摄取的定量将在数据分析部分讨论。 (图1和图2)


      图1.用100μg/ml 10kDa FITC标记的葡聚糖分子装载4小时的未转导和未处理的MDDC的实施例。在左图(亮视野)上可以看到盖玻片上的细胞。在右面板上,显示400nm的一个Z-堆叠部分,其中细胞的细胞核用DAPI(蓝色)染色,葡聚糖分子可以用绿色可视化。


      图2.通过共聚焦显微镜检查HIV-1内化的实施例 MDDCs未转导(上图)或用针对DNM2的shRNA转导(下图),用50ng HIV-1(绿色点)。明场可以在左面板上看到。在右面板上,显示400nm的一个Z-堆叠部分,其中细胞的核用DAPI(蓝色)染色,红色的肌动蛋白细丝,在鬼笔环肽染色后和捕获的HIV-1以绿色。

数据分析

  1. 通过流式细胞术分析HIV-1或葡聚糖捕获(图3)
    1. 首先,门必须设置在活细胞上,通过排除已经结合活/死染色的细胞。重要的是MDDC是高度分化的,如通过DC-SIGN的高表达和CD14的低表达所评估的。 (图3A)
    2. 单核细胞通常是CD14高和DC-SIGN低。
    3. 可以通过比较平均荧光强度(MFI)与阴性对照并确定样品之间的内在化比率来计算HIV-1或内含的葡聚糖的量(图3B)。
    4. 建议通过使用不同的稀释度和不同的孵育时间来评估HIV和葡聚糖捕获,以获得代表性的和更准确的曲线。
    5. 每个样品通常一式三份进行分析,并且用来自至少3个独立的健康献血者的细胞进行实验。
    6. 样品之间MFI差异的统计分析可以使用Holm-Sidak多重比较检验进行单向ANOVA(NS,不显着; * P <0.05; ** P <0.01; ***

      <0.001; **** P <0.0001)。


      图3.通过流式细胞术分析HIV-1和葡聚糖捕获 A.分析MDDC的门控策略:左图,基于大小(FSC)和粒度(SSC)选通MDDC;对活化分化的MDDC(DC-SIGN +; dapi-)的右面板门控。 B.在装载有HIV-1(上图)或葡聚糖(下图)的MDDC上门控后产生的直方图的实例。在灰色中,P24的同种型对照用于评估由scramble shRNA相对于TSPAN7 shRNA转导的MDDC捕获的HIV-1的量。对于葡聚糖内化,灰色直方图表示阴性对照,其中细胞保持在4℃以防止内化。

  2. 通过定量PCR(QPCR)分析HIV-1捕获(图4)
    1. 在逆转录后,通过使用用于GFP(编码在HIV基因组内部)的引物的定量PCR检测HIV-1RNA的量,并归一化为GAPDH。
    2. 为了以最准确的方式评估数量,每个实验应包括使用GFP引物和GAPDH引物的标准曲线。
    3. 然后使用标准曲线来确定每个样品中内化的HIV的量,并将其与阴性对照(在4℃下没有进行病毒或捕获实验)进行比较。
    4. 可以在不同样品之间比较标准化的HIV-1数量,以评估HIV捕获的增加或减少(图4)。
    5. 由于可能存在于不同血液供体之间的实验变化,优选不比较来自不同供体的样品,而是在趋势方面寻求供体之间的一致性。实验应该一式三份,并在至少五个不同的献血者。
    6. 可以使用Holm-Sidak多重比较检验,根据单因素方差分析(one-way ANOVA)(NS,不显着; * P <0.05; P <0.01; ***

      <0.001; ****

      <0.0001)

      图4.通过定量PCR分析HIV-1捕获。在HIV捕获实验后通过定量PCR分析直方图的实例。用scramble shRNA转导的MDDC,空载体或针对TSPAN7的shRNA加载HIV-GFP 4或24小时。通过针对GFP的PCR分析捕获的HIV-1的量,并与scramble shRNA(任意设置为1)之后捕获的量进行比较。

  3. 通过共聚焦显微镜分析HIV-1或葡聚糖捕获(图5)
    1. 对于通过共聚焦显微镜的葡聚糖内化的定量,我们使用鬼笔环肽染色(对于丝状肌动蛋白)以鉴定每个细胞的外部边界。
      1. 使用ImageJ软件,为每个Z-堆叠收集每个像素的葡聚糖的最大强度。
      2. 然后获得葡聚糖的总强度并归一化为所分析的体积。
      3. 积分强度可以通过取每个强度大于背景(强度≥200)的所有体素的总和,然后乘以强度和求和来计算。
      4. 通过计数大于背景的所有体素获得体积。
      5. 浓度是积分强度除以体积(任意单位)(图5)。
      6. 对于每个样品,我们建议计算至少100个细胞的葡聚糖的浓度并且用至少3个不同的人血液供体重复实验。未配对的 t 测试可用于确定统计显着性
    2. 关于HIV-1内化的定量,使用共聚焦显微镜获得400nm Z-堆叠,每个细胞平均具有20个Z-堆叠。
      1. 基于其与皮层肌动蛋白屏障(鬼笔环肽染色)相比,每个HIV-1颗粒(绿点)可以手动计数为内化。
      2. 基于电子显微镜数据,估计每个HIV-1聚集体存在5个HIV病毒颗粒的平均值。
      3. 对于每个细胞,因此可以计算HIV-1的总量,并且分析每个样品至少100个细胞的平均值。未配对的 t 测试可用于确定统计显着性

        图5.通过共聚焦显微镜检查的葡聚糖捕获的分析。用针对TSPAN7的scramble shRNA或shRNA转导的MDDC用10kDa(左图)或70kDa葡聚糖分子(右图)培养4小时, 。然后如上所述测定由每个MDDC内化的葡聚糖的浓度。简言之,浓度是葡聚糖染色的积分密度除以体积,并且在y轴上以任意单位显示。对于每个条件,每个点表示不同的单元格,平均值以±SEM显示

笔记

  1. 一般来说,当使用人类原代细胞时,您需要确保具有内部控制,以便能够标准化数据和比较来自不同献血者的数据。也可以观察到捐赠者对捐赠者的变化,并且增加不同的人类献血者的数量通常有助于更好地分析数据。
  2. 我们建议始终检查MDDC的成熟状态。该方案中描述的实验使用未成熟的MDDC进行。一旦成熟,就HIV-1捕获和葡聚糖内化而获得的结果是不同的。通过流式细胞术观察CD86和CD80分子可以确定成熟状态。不成熟的MDDC应该是CD80和CD86低。
  3. 所有3种技术测量MDDC的HIV-1内化。组合3种不同的方法增加了检测甚至捕获和内化中的小差异的机会,并且增加结果的鲁棒性。为了检测HIV-1内化的变化,流式细胞术可能不是最好的方法,因为它似乎没有像QPCR或共聚焦显微镜检测小量的艾滋病毒内部化敏感。定量PCR可能是最快和最简单的方法,但不允许直接比较HIV-1和葡聚糖内化,并且任何伪影污染可能潜在地偏差数据。最苛刻的技术可能是共焦显微镜,但允许在不同的样品之间的HIV-1和葡聚糖内化的最严酷的比较。

食谱

  1. D10媒介
    DMEM
    10%胎牛血清(FBS,热灭活) 0.1 mM MEM非必需氨基酸
    6mM L-谷氨酰胺 1mM MEM丙酮酸钠 100 U/ml青霉素
    100μg/ml链霉素 50μg/ml庆大霉素
  2. MACS缓冲区
    DPBS
    0.5%牛血清白蛋白(BSA)
    1mM EDTA
  3. DC介质
    RPMI
    10%FBS(热灭活) 10mM Hepes(Hyclone)
    55μMβ-巯基乙醇 6mM L-谷氨酰胺 100 U/ml青霉素
    100μg/ml链霉素 50μg/ml庆大霉素
  4. 2×PHEM缓冲液(500ml)
    18.14克PIPES
    6.5 g HEPES
    3.8 g EGTA
    0.99g MgSO 4 用10N KOH调节pH至7.0
  5. DABCO-PVA
    2.5%Dabco
    10%聚乙烯醇(PVA) 5%甘油 25mM Tris缓冲液,pH8.7

致谢

我们感谢Alice F. Liang,Michael Cammer和NYULMC OCS显微镜核心为光学显微镜提供的服务;林文林技术帮助;和Jarrod Johnson,Nicolas Manel,他们的批评性建议。这项工作得到了来自EMBO,癌症研究所和Philippe基金会(M.M.M.)的研究金的支持;由霍华德休斯医学研究所(D.R.L.)和海伦和马丁·金梅生物和医学中心(D.R.L.);和国立卫生研究院(R21AI084633)(D.R.L.)和NCRR(S10RR023704-01A1)的赠款。

参考文献

  1. Dustin,ML,Starr,T.,Varma,R.and Thomas,VK(2007)。  支持用于研究免疫突触的平面双层。 Curr Protoc Immunol 第18章:单位18 13.
  2. Hubner,W.,Chen,P.,Del Portillo,A.,Liu,Y.,Gordon,RE and Chen,BK(2007)。  1型人类免疫缺陷病毒(HIV-1)的序列Gag定位和寡聚化用复制能力的荧光标记的HIV的活共聚焦成像监测-1。 J Virol 81(22):12596-12607。
  3. Menager,MM和Littman,DR(2016)。  肌动蛋白动力学调节树突细胞介导的HIV-1转移到T细胞。细胞 164(4):695-709。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ménager, M. M. and Littman, D. R. (2016). Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs). Bio-protocol 6(22): e2004. DOI: 10.21769/BioProtoc.2004.
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