Cellular Activation Measurement for HIV

Cellular activation was assessed by measurement of HLA-DR and CD38, similar to previous studies (12 (link), 36 (link), 37 (link)). Staining for flow cytometry was performed both extracellularly and intracellularly. The extracellular staining cocktail consisted of LIVE/DEAD Amcyan fixable dye (Thermo Fisher Scientific, Waltham, MA, USA), anti-CD3-APC-H7, anti-CD4-BV605, anti-CD8-BV655, anti-CD14-Pacific blue (all from BD Biosciences, Franklin Lakes, NJ, USA), and anti-CD19-pacific blue (Biolegend, San Diego, CA, USA). The intracellular staining cocktail consisted of anti-CCR5-APC, anti-HLA-DR-PerCP-CY5.5 (all from BD Biosciences, Franklin Lakes, NJ, USA), anti-CD38-PE-CY7 (Biolegend, San Diego, CA, USA) and anti-p24-FITC (Beckman Coulter, Brea, CA, USA). PBMCs were collected at two time-points: day 3 (48 h post stimulation and prior to HIV infection) and day 5 (48 h post infection).