High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

NOTE: Procedures for cloning, transformation and sequencing are discussed elsewhere^18,19. The protocols herein begin from the transfection of the mammalian expression vectors (Figure 3).

1. Transfection of HEK293T Cells with Dual Color Reporter Construct

1. Cultivate HEK293T cells in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL) in a 5% CO₂ incubator at 37 °C. After thawing, passage HEK293T cells 2-3 times before using them in transfection experiments. This would give the cells time to recover from the thawing procedure.
   CAUTION: Mycoplasma contamination of cell cultures remains a serious problem. Good laboratory practice and routine testing of cell cultures are essential to decrease the risk of mycoplasma contamination and avoid diffusion²⁰.
2. Split the cells 1:2 one day before seeding and transfer them into fresh DMEM medium. Cultivate the cells for 24 h in a 5% CO₂ incubator at 37 °C.
3. The day before transfection, remove the medium from the flask by aspiration and wash the cells with Dulbecco’s phosphate buffered saline (DPBS) solution free of calcium (Ca²⁺) and magnesium (Mg²⁺) to remove traces of serum.
4. Dispense 5 mL of trypsin-EDTA solution (0.05% – 10 mM in PBS) into the culture vessel and place it at 37 °C in a 5% CO₂ incubator for 2 to 5 min.
5. When the cells are detached, add 5 mL of DMEM supplemented with 10% (v/v) FBS to further inhibit the trypsin activity which may damage the cells.
6. Resuspend gently by pipetting the cell suspension up and down to break up the clumps.
7. Dilute the cells 1:10 in trypan blue stain (0.4%).
8. Count the live cells that do not take up trypan blue using a microscope (10x objective) and a haemocytometer.
9. Calculate the number of viable cells per milliliter by taking the average of the cell count and multiplying it by 10,000 and by the dilution factor (1:10) from the trypan blue stain.
10. Plate 2 x 10⁴ cells in 100 μL of DMEM medium supplemented with 10% FBS without antibiotics in a 96-well flat-bottom plate for 24 h.
11. For each well, dilute 400 ng of pLTR.gp140/EGFP.RevD38/DsRed, 20 ng of pCMV-Rev^NL4.3 and 100 ng of pCMV-Tat101^AD8-Flag DNA in 50 μL of serum free medium. Mix gently. For experiments without Tat, use a matched empty Tat vector pcDNA3.1 (-).
    NOTE: The use of endotoxin-free DNA is highly recommended. For that, prepare endotoxin-free DNA using a midi or maxiprep nucleic acid purification kit. Determine the DNA purity by measuring the 260/280 OD ratio, which should be between 1.7 and 1.9.
12. Mix the lipid reagent gently before use, then dilute 0.65 μL in 50 μL of serum free medium. Mix gently and incubate for 5 min at room temperature.
13. Combine the diluted DNA with the diluted lipid reagent.
14. Mix gently and incubate for 20 min at room temperature. The solution may appear cloudy.
    NOTE: Proceed to step 1.15. within 30 min.
15. Dispense 100 μL per well of the lipid reagent-DNA complexes drop-wise on top of the cells.
16. Mix gently by rocking the plate back and forth.
17. Incubate the plate for 5 h in a 5% CO₂ humidified incubator at 37 °C.
    1. Each reaction mix is sufficient for a single well (96-well) transfection. Adjust the amount and volume of components according to the number of drug and combination that would be tested, and accounts for pipetting variations. All conditions are usually tested in triplicates.
    2. Transfect additional wells with 100 ng of CMV-driven EGFP and DsRed-Express DNA plasmid for compensation purposes.

2. Treatment of Transfected HEK293T Cells with Latency Reversing Agents

NOTE: Prior to each assay, determine the physiological condition of each LRA by measuring the viability of the cells after exposure to high and low dose of the drug with cell proliferation assay.

1. Dilute each LRA to the appropriate working concentration with growth medium (e.g., 1 μM for JQ1(+)).
   CAUTION: Reconstitute the lyophilized drugs with the appropriate solvent to a concentration of 10 mM. Store all stock LRAs at -80 °C in a single use aliquot (5 μL each) in order to avoid repeated freezing-thawing cycles.
2. Carefully aspirate transfection medium with a multichannel and replace with 100 μL medium containing appropriate LRA (Table 1). Add medium very gently alongside the wall of the well to avoid detaching the transfected HEK293T cells, which are known to easily detach from the culture plate surface.
3. Incubate the plate for 48 h in a 5% CO₂ humidified incubator at 37 °C.

3. Staining of Transfected Cells with Fixable Viability Dye for Flow Cytometry Analysis

CAUTION: Removing dead cells and debris is essential to eliminate false positives and to obtain results of the highest quality.

1. Detach the cells into the media using 100 μL of phosphate-buffered saline (PBS) per well and by gently pipetting up-and-down (~5 times) with a multichannel, while avoiding frothing of the media. If needed, detach the cells from the plate using 35 µL per well of trypsin-EDTA solution (0.05% – 10 mM in PBS) and incubating 5 min at 37 °C, before neutralizing with medium containing serum.
2. Transfer the cells to a 96-well V-bottom plate.
3. Spin the cells for 5 min at 500 x g at 4 °C, then carefully aspirate the medium/PBS without touching the cells.
4. Wash the cells at least 1 time with 200 μL of protein/serum free PBS.
5. Centrifuge at 500 x g for 5 min at 4 °C, then discard the supernatant by tilting the plate and removing the wash buffer without touching the cells.
6. Prepare a working solution of fixable viability dye by diluting the viability dye 1:1000 in protein/serum free PBS. Prepare 50 µL of the diluted stain per well.
   NOTE: Viability dyes are available in a range of colors suitable for use with blue, red and violet lasers. Prepare a stock solution of fixable viability dye by resuspending one vial of the lyophilized dye (component A) with 50 μL anhydrous DMSO (component B). Store at -20 °C in a single use aliquot (1 μL each), protected from the light.
7. Add 50 μL of the diluted viability dye to each well and mix the cells by pipetting up and down with a multichannel.
8. Stain for 10-15 min at 4 °C, protected from the light.
9. Wash 1-2 times with 150 μL of wash buffer (PBS with 1% bovine serum albumin and 2 mM EDTA).
10. Centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
11. Fix the cells with 100 μL of freshly prepared 1% formaldehyde in wash buffer for 10-15 min at 4 °C in the dark.
    CAUTION: Formaldehyde is very toxic. Prepare 1% formaldehyde solution in a fume hood to avoid inhalation while wearing gloves and safety glasses for protection.
12. Wash the cells 1-2 times with 100 μL of wash buffer.
13. Centrifuge at 500 x g for 5 min at 4 °C. Discard the supernatant.
14. Resuspend the cell pellet in 70 μL of wash buffer.
    NOTE: The protocol can be paused here. Fixed cells could be stored at 4 °C in the dark for analysis the next day on the flow cytometer.

4. EGFP and DsRed Measurements by Flow Cytometry and Data Analysis

NOTE: Analyze HIV transcription (% EGFP) and splicing (% DsRed) on a flow cytometer. Filter the sample before the run with a 70 μm cell-strainer or 100 μm nylon mesh to avoid clogging up the nozzle.

1. Start up the cytometer and computer at least 10 min prior to use to ensure lasers warm up.
2. Prior to running samples and commencing data acquisition, fill the sheath tank with 0.9% saline solution and ensure that a sodium hypochlorite tablet is added to the waste tank.
3. Check the flow cell for air bubbles.
4. Verify that the detectors and filters are appropriate for the experiment.
   NOTE: For EGFP, use of blue laser (488 nm) and 530/30 bandpass, while DsRed Express is optimally detected using the yellow laser (561 nm) and 610/20 bandpass. A blue laser (488 nm) and 610/20 bandpass can also be used to detect DsRed expression.
5. Check the cytometer performance and sensitivity across detectors by running calibration beads.
6. Adjust the forward (FSC-A) and side (SSC-A) scatter voltages with unstained sample so that the main population is on-screen and clearly discernable.
   NOTE: FSC (Forward-scattered light) is a measurement of light diffraction in a flat angle, which depends on the volume of the cell (cell surface area or size), while SSC (side-scattered light) is a measurement of light diffraction in a right angle, which is proportional to cell granularity and internal complexity.
7. Perform manual or automatic compensation by using the single-stained samples ensuring minimal spillover of EGFP+ population into the DsRed detector and vice-versa.
   CAUTION: For compensation purposes, use a sample of cells expressing each of the single color fluorescent protein as well as cells singly stained with the fixable viability dye. Do not use FITC and PE compensation beads for EGFP and DsRed fluorescent proteins compensations due to different spectral characteristics. Compensation controls should be bright enough to resolve positive and negative population.
8. Create plots and set the gates using fluorescence minus one (FMO) controls.
9. Acquire and record a minimum of 10,000 viable cell events per sample. Run samples at medium or low speed to avoid doublets passing through the laser intercept. Use pulse geometry gating such as SSC-A versus SSC-H to eliminate doublets. Check the stability of the run by plotting time versus SSC-A to see how even the flow is during the run.
10. At the end of the run, clean the flow cytometry fluidics with concentrated cleaning agents then water for 5 min each.
11. Shutdown the system, discard the waste and re-fill the sheath tank after acquisition.
12. Analyze the data using a flow cytometry data analysis software. Exclude cell debris and clump (doublets) based on forward and side scatter, then eliminate the dead cells using the viability dye stain (negative population = live cells). Identify the cells expressing EGFP and DsRed (Figure 4), as well as the percentage of spliced product DsRed/(DsRed + EGFP) (Figure 5).
13. Determine the effect of the LRA on HIV transcription and splicing by comparing untreated cells and the cells exposed to individual or a combination of LRAs (Figure 5).