Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines

Materials and Reagents

1. A2 and A72 J-Lat cell culture
   1. 75 cm² tissue culture flask (Corning, Falcon^®, catalog number: 353110 )
      
   2. Tips
      0.1-10 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-509 )
      
   3. A2 and A72 J-Lat cells (Jordan et al., 2003)
      
   4. RPMI (Mediatech, catalog number: 10-040-CV )
      
   5. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
      
   6. L-glutamine (Mediatech, catalog number: 25-005-Cl )
      
   7. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
      
      
2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
   1. 96-well V-bottom tissue culture plates and lids (Thermo Fisher Scientific, Thermo Scientific ^TM, catalog numbers: N249570 and N163320 )
      
   2. Tips
      0.1-10 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-681-440 )
      1-200 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-502 )
      101-1,000 µl (Fisher Scientific, Fisherbrand^TM, catalog number: 02-707-509 )
      
   3. RPMI (Mediatech, catalog number: 10-040-CV )
      
   4. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-106 )
      
   5. L-glutamine (Mediatech, catalog number: 25-005-Cl )
      
   6. 100x penicillin/streptomycin (Mediatech, catalog number: 30-002-Cl )
      
   7. TNFα (PeproTech, catalog number: 300-01A )
      
   8. JQ1 (Cayman Chemical, catalog number: 11187 )
      
   9. Prostratin (Sigma-Aldrich, catalog number: P0077 )
      
   10. Suberoylanilide hydroxamic acid (SAHA) (Sigma-Aldrich, catalog number: SML0061 )
       
   11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
       
   12. 1x PBS (Mediatech, catalog number: 21-031-CV )
       
   13. MACSQuant Running buffer (Miltenyi Biotech, catalog number: 130-092-747 )
       
   14. ApoTox-Glo^TM Triplex Assay (Promega, catalog number: G6320 )
       
   15. RPMI medium (see Recipes)
       
   16. TNFα stock solution (see Recipes)
       
   17. JQ1 stock solution (see Recipes)
       
   18. Prostratin stock solution (see Recipes)
       
   19. Suberoylanilide hydroxamic acid (SAHA) stock solution (see Recipes)
       

Equipment

1. Pipette
   
2. Biosafety cabinet level 2
   
3. CO₂ tissue culture incubator (Thermo Electron, model: Forma^TM Steri-Cult^TM CO₂ Incubators , catalog number: 3307)
   
4. Tabletop centrifuge (Beckman Coulter, model: Allegra X-14R ) for 96-well plates
   
5. MACSQuant VYB FACS analyzer (Miltenyi Biotech, model: MACSOuant^® VYB, catalog number: 130-096-116 )
   
6. SpectraMax MiniMax^TM 300 Imaging Cytometer (Molecular Devices, model: SpectraMax MiniMax 300 )
   

Software

1. FlowJo 9.9 or never (Tree Star)

Procedure

1. A2 and A72 J-Lat suspension cell culture
   1. A2 and A72 cells are cultured in RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin) in 75 cm² tissue culture flasks in 40 ml medium. The flasks are placed horizontally to increase the surface area in which the cells are grown.
      
   2. Cells are split every 1-2 days and should be kept at a concentration of 2 x 10⁵/ml-1.5 x 10⁶/ml.
      
   3. One day before drug treatment split A2 or A72 cells to 5 x 10⁵/ml.
   
   
2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry
   1. Count the cells and adjust cell number with new media. About 2 x 10⁵/well of A2 or A72 cells are plated in 96-well tissue culture plates in 195 µl RPMI medium (supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin). For each condition perform three replicates.
      
   2. Cells are stimulated with 0.016-10 ng/ml of TNFα, 100 nM-1 µM JQ1, 100 nM-2.5 µM Prostratin, 330 nM-2.5 µM SAHA, DMSO control, or left untreated. If treated with TNFα, use PBS or water as control, depending on what was used as diluent. All drugs and controls are pipetted in 5 µl to reach a total volume of 200 µl/well. Mix cells and drugs by pipetting up and down at least 3 times.
      
   3. Culture in a CO₂ incubator at 37 °C for 18-48 h.
      
   4. Take the plate from the incubator and centrifuge at room temperature for 2 min at 1341 x g (2,400 rpm) (Tabletop centrifuge, Beckman).
      
   5. Then proceed directly to flow cytometry analysis. Cells do not have to be washed or fixed.
      Notes:
   1. For flow cytometry, MACSQuant VYB FACS analyzer was used to run 96-well plates. However, other flow cytometers such as Calibur or LSRII are also suitable for these experiments.
      
   2. If the analysis of cell viability by forward scatter (FSC) vs. side scatter (SSC) is not sufficient for accurate assessment of drug toxicity in drug studies, one of a variety of dyes/stains; for example 7AAD, Propidium iodide or one of the Zombie viability dyes can be used according to manufacturer’s instructions in the flow cytometric analysis in this protocol.
      
   6. Viability, cytotoxicity and apoptosis was measured with ApoTox-Glo^TM Triplex Assay (Promega) according to manufacturer’s instructions using a SpectraMax MiniMax^TM 300 Imaging Cytometer (Figure 1).
      
      
      Figure 1. Measurement of Viability, Cytotoxicity and Apoptosis of drug treated cells. ApoTox-GloTM Triplex Assays (Promega) were performed in drug-treated A72 J-Lat cells. A. Cytotoxicity and Viability; B. Apoptosis; All measurements were repeated at least three times and average of three technical replicates (± SD) is shown.
      

Data analysis

Analysis of HIV-1 LTR transcriptional activation by flow cytometry (Figure 2)

1. First, set the gate on live J-Lat cells. Cell viability is monitored by forward (FSC-Area) and side scatter (SSC-Area) analysis (Figure 2A).
   
2. Gate on singlets (FSC-Height vs. FSC-Area) (Figure 2B).
   
3. Set the gate on SSC-Area and GFP/FITC-Area to identify the amount of GFP⁺ cells (Figure 2C).
   
4. Each sample is usually analyzed in triplicate and the experiment is performed with cells coming from at least 3 independent experiments (Figure 3). Three replicates are averaged by calculating (GFP⁺ cells Experiment 1 + GFP⁺ cells Experiment 2 + GFP⁺ cells Experiment 3)/3. Also calculate standard deviation (STDEV) for error bars.
   
   
   Figure 2. Analysis of HIV-1 LTR transcriptional activation by flow cytometry. Gating strategy to analyze A2 or A72 J-Lat cells: A. gating on live J-Lat cells based on size (FSC-Area) and granularity (SSC-Area); B. singlets gate (FSC-Height vs. FSC-Area); C. gating on GFP⁺ J-Lat cells (SSC-Area vs. GFP-FITC-Area).
   
   
   Figure 3. HIV-1 LTR transcriptional activation by flow cytometry. Typical results obtained for 18 h treatment with TNFα, with dose dependent response using A2 J-Lat cells. Average for percentage of GFP⁺ cells from three replicates (± SD) is shown.
   

Recipes

1. RPMI medium
   RPMI supplemented with,
   10% FBS
   1% L-glutamine
   1% penicillin/streptomycin
   Store at 4 °C
   
2. TNFα stock solution
   100 ng/μl in sterile water
   Store at -80 °C
   
3. JQ1 stock solution
   10 mM in DMSO
   Store at -80 °C
   Note: Avoid repeated freeze-thaws!
   
4. Prostratin stock solution
   5 mM in sterile water
   Store at -20 °C
   
5. Suberoylanilide hydroxamic acid (SAHA) stock solution
   10 mM in DMSO
   Store at -20 °C
   

Acknowledgments

We thank Marielle Cavrois and the Flow cytometry core for the service provided for flow cytometry. This publication was made possible with the help from the University of California, San Francisco–Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027763). This research was supported as part of the amfAR Institute for HIV Cure Research, with funding from amfAR grant number 109301. Further, we gratefully acknowledge support from the California HIV/AIDS Research Program (Award number: F13-GI-316) to D.B., and grant support from the CARE Collaboratory (U19 AI096113) and the NIH (RO1 AI083139 and RO1 DA043142) to M.O. This protocol was adapted from previous work: ‘BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism’ (Boehm et al., 2013).

References

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