Cd3 pacific blue ucht1

CD4⁺ T-cells were isolated from total PBMCs by negative selection using EasySep Human CD4 T Enrichment columns (StemCell, Cat #19052). Purity of CD4 was typically >98% as determined by surface staining of CD4⁺ T-cells with CD3-Pacific Blue (UCHT1) and CD4-Alexa Fluor 700 (RPA-T4), (both from BD) and FACS analysis (BD LSRII analyser). Purified CD4⁺ T-cells were stimulated with plate-coated anti-CD3 antibody (0.5μg/ml) and soluble anti-CD28 antibody (0.5μg/ml) (both from BD Biosciences, Cat ##555329, 5555726 respectively) for 48 h in the presence or absence of 500 ng IL-32α (BioLegend, Cat # 551004) or IL-32γ (R&D Cat # 4690-IL-025/CF). Secreted cytokines were measured from the supernatant of activated cells using the LEGENDplex™ Human Th Cytokine Panel (13-plex) (BioLegend, Cat # 740001) according to manufacturer’s directions. Infections of PBMCs stimulated with PHA and IL-2 (0.25μg/ml, and 100 units/ml, respectively) or resting cells were carried out using 50 ng of the laboratory strain HIV-BaL per 10⁶ cells by Spinoculation61 (link) (2 hours/Room temperature). Following infection, PBMCs were washed twice by PBS and centrifuged at 1500 rpm for 5 min then were re-suspended in RPMI-1640 medium supplemented with 10% FBS. Cells were kept in culture for 48 h before collection of supernatants and cells for cytokine measures.