Optimization of J-Lat 6.3 cells activation by TNFα/Aza-CdR. HIV latently infected J-Lat 6.3 cells that contain the GFP reporter gene were (A) treated with Aza-CdR (2.5 μM) and TNFα (10 ng/ml) for 48 h, (B) were treated with Aza-CdR plus TNFα and then washed at 24 h, (C) treated with Aza-CdR followed by addition of TNFα after 24 h of Aza-CdR treatment, and (D) treated with Aza-CdR plus TNFα for 24 h, followed by the addition of fresh Aza-CdR. Cells were cultured in RPMI (Atlanta Biologicals, Lawrenceville, GA) with 10% FBS (Invitrogen, Carlsbad, CA). Two million cells were seeded with 3 ml medium in six well plates and treated with Aza-CdR (2.5 μM), TNFα (10 ng/ml) or both compounds combined. After 48 h cells were transferred to a 15 ml conical tube and spun down at 1400 rpm for 7 min. The supernatant was discarded and cell pellets were resuspended and were washed twice with chilled 1× PBS and fixed in 2% formaldehyde for FACS analysis using a FACScalibur cytometer (BD Biosciences, San Jose CA) and analyzed with Cell Quest Pro software (BD Biosciences, San Jose CA). GFP positive cells from the live population, defined by forward versus side scatter gating, were quantified. 20.000 events per treatment condition were analyzed. Results are the mean ± standard deviation (SD). The TNFαAza-CdR/TNFα (FI-Aza) ratio was calculated and used to evaluate the optimum time of activator addition.