The overall goal of this procedure is to assess in vitro, the impact of latency reversing agents on HIV mRNA processing. The protocol provide a simple efficient and reliable method for assessing, simultaneously the effect of LRA on HIV transcription and splicing. This technique provided an insight on the ability of LRAs to induce virus reactivation and clearance of the latent reservoir.
After cultivating cells, place 20, 000 of them in 100 microliters of DMEM medium, supplemented with 10%FBS, and without antibiotics in the wells of a 96-well flat bottom plate for 24 hours. The next day dilute the dual color reporter construct. Tat and Rev DNA in 50 microliters of serum free medium, and mix gently.
Gently mix the lipid reagent, and then dilute 0.65 microliters of it per reaction in 50 microliters of serum free medium. Mix gently, and incubate at room temperature for five minutes. Combine the diluted lipid reagent with the diluted DNA and gently mix.
Incubate at room temperature for 20 minutes. Then, dispense 100 microliters of this lipid reagent DNA complex drop wise, on top of the cells in each well. Rock the plate gently back and forth to mix.
And incubate the plate in an humidified incubator, at 37 degrees celsius with 5%carbon dioxide for five hours. After this, transfect additional wells with 100 nanograms of CMV driven EGFP and DsRed express DNA plasmid for compensation purposes. To begin, dilute each latency reversing agent to the appropriate working condition with growth medium.
Using a multi-channel pipe head, carefully aspirate the transfection medium, and replace it with 100 microliters of medium containing the appropriate latency reversing agent. Incubate in a humidified incubator at 37%celsius with 5%carbon dioxide for 48 hours. To begin staining the cells, use a multi-channel pipe head to add 100 microliters of PBS to each well.
Gently pipe head up and down approximately five times. While making sure to avoid frothing to detach the cells into the media. Next, transfer the cells into the wells of a 96-well V-bottom plate.
Centrifuge at 500 times gravity and at four degrees celsius for five minutes. Aspirate the medium and PBS without touching the cells. Wash the cells at least once with 200 microliters of protein and serum free PBS.
Centrifuge at 500 times gravity and at four degrees celsius for five minutes. Tilt the plate to discard the supernatant and remove the wash buffer without touching the cells. After this dilute the fixed viability dye in protein and serum free PBS at a ratio of 1 to 1000, to prepare a working solution.
Add 50 microliters of the diluted dye to each well, and pipe head up and down to mix. Incubate at four degrees celsius. While protected from light using foil for 10 to 15 minutes.
Then wash the cells once or twice with 150 microliters of wash buffer. After this, centrifuge at 500 times gravity, and four degrees celsius for five minutes, and discard the supernatant. To fix the cells, add 100 microliters of freshly prepared 1%formaldehyde and wash buffer.
And incubate at four degrees celsius while protected from light for 10 to 15 minutes. After this, wash the cells once or twice with 100 microliters of wash buffer. Centrifuge at 500 times gravity and at four degrees celsius for five minutes.
Discard the supernatant, and resuspend the cell pellet in 70 microliters of wash buffer. After checking the cytometer settings performance and sensitivity by running calibration beads, adjust the forward and side scatter voltages with unstained sample, so that the main population is on screen and clearly discernible. Perform manual or automatic compensation by using the single stained samples, ensuring minimal spill over of EGFP positive population into the DsRed detector and vice versa.
Using the fluorescence minus one controls, create plots and set the gates. Next, adjust the forward and side scatter voltages with unstained sample, so that the main population is on screen and clearly discernible. Acquire and record a minimum of 10, 000 viable cell events per sample.
After this, use flow cytometry data analysis software to analyze the data as outlined in the text protocol. Representative results for the expression of HIV-1, unspliced and spliced products, after treatment with the bromodomain inhibitor JQ1, show that both JQ1 positive and Tat, significantly increase the percentage of cells expressing EGFP. Which is indicative of unspliced transcripts.
JQ1 positive is also seen to significantly increase the percentage of cells expressing DsRed. And increase the proportion of spliced product to similar levels as Tat. This confirms the ability of JQ1 positive to turn on HIV transcription and splicing.
However, treatment with a stereoisomer control JQ1 negative, abolishes the JQ1 positive effect on HIV transcription and splicing. Once mastered, this method can be carried out using drugs individually or in combination. Testing for their ability to synergize efficiently.
Which would lead to a lower dose of the drug and toxicity. After each development, this technique paved the way for testing efficient drug combinations in primary models of latency in ex vivo patient samples.
