Live dead aqua blue

This method has been explained in detail elsewhere (Mujib et al, 2017 (link)). Briefly, negatively isolated CD4⁺ T cells from cryopreserved PBMCs (according to the manufacturer’s protocol, EasySep Human CD4⁺ T-cell isolation kit; STEMCELL Technologies Canada Inc.) were activated on plate bound 1 μg/ml each of anti-human CD3 and anti-human CD28 (clones OKT3 and 28.2, respectively; BioLegend), and 50 IU/ml recombinant human IL-2 (rhIL-2; Roche Diagnostics) for 3 d before infecting with HIV-1 NL4-3 provirus. The killing assay was set up 3 d after infection with CD8⁺ T cells and HIV-1–infected CD4⁺ T cells at 1:1 ratio with 100,000 of each cell in the presence of 10 IU/ml rhIL-2 and the various inhibitors, if present. After overnight killing, the cells were stained with Live/Dead Aqua Blue (Life Technologies) and surface-stained with anti-human CD3 BV605, anti-human CD8 BV711, and anti-human CD4 APC (all from BioLegend). Afterwards, the cells were permeabilized and stained with anti–HIV-1 Gag p24 antibody Kc57-FITC (Beckman Coulter) to detect intracellular HIV-1 Gag protein. Samples were run on modified five laser BD LSR Fortessa X-20. Data were analyzed on FlowJo (FlowJo).

Cryopreserved PBMCs were thawed, washed, and stained with Live/Dead Aqua Blue (Life Technologies), followed by surface staining with antihuman CD3 BV605, anti-human CD4 BV650, anti-human CD8 BV711, anti-human CD27 PerCP-eF710, CD45RO PE-CF594 (BD Biosciences), anti-human CD98 PE (BD Biosciences), anti-human CD71 APC, anti-human PD-1 PE-Cy7, and anti-human T-Bet BV421 (all from BioLegend unless otherwise noted). Anti-human CD14, anti-human CD16, and anti-human CD19 all tagged with V500 (BD Biosciences) were added in dump. For median fluorescence index determination, manual target channeling was performed with 1× beads (SpiroTech). Samples were run on modified 5 laser BD LSR Fortessa X-20. Data were analyzed on FlowJo (FlowJo).