Quantifying Latent HIV Infection in CD4+ T-cells

Latent infection in the sorted, non-proliferating CD4⁺ T-cells (eFluor670^hiEGFP⁻) was determined by comparison of stimulated with un-stimulated T-cells sorted from APC-T-cell co-cultures (control). 1x10⁵ sorted CD4⁺ T-cells were stimulated with immobilized anti-CD3 (7 μg/ml; Beckman Coulter), in RF10 media supplemented with CD28 (5 μg/mL; BD Biosciences), IL-7 (50 ng/mL; Sigma, St Louis, MO, USA), IL-2 (5U/mL; Roche), with (post-integrated latency) or without (total latency: pre- and post-integrated latency) integrase inhibitor L8 (1 μM; Merck, White House Station, NJ, USA). The concentration of L8 was determined previously by titration of L8 in phytohaemagglutinin (PHA; 10 μg/mL) activated PBMC infected with R5-EGFP virus at an MOI of 0.5, same concentration usedin co-cultures, and showed productive infection was completely blocked at 1 μM. This concentration used for all subsequent experiments. Cells were harvested after 72 h of stimulation and EGFP expression was quantified on the FacsCalibur (BD BioSciences).
In some experiments PHA (10ug/mL) and IL-2 (10 U/mL) stimulated feeder PBMC were used to activate T-cells as a measure of inducing virus replication form latency, as described previously [14 (link)].