Anti cd4 pe dazzle594

Non-activated bbPBMCs seeded at 4 X 10⁶ were treated in vitro with the indicated ligands for 7 days in full RPMI containing 10% (v/v) cs-FCS, 2mM L-glutamine, 0.1 mg/mL sodium pyruvate, 100 IU/mL penicillin, 100 mg/mL streptomycin and 30 U/mL IL-2, while hPBMCs were thawed and rested overnight in full RPMI in an incubator at 37°C. Cells were washed with 1 X PBS containing 1% cs-FCS and subsequently stained with anti-human CD3 FITC (300306), anti-CD4 PE-DAZZLE 594 (357412), anti-CD8 PE/Cy5 (300910), anti-CD14 APC (325608), anti-CCR5 PE (359106), anti-CD69 PE/Cy7 (310912) and the viability dye, ZOMBIE NIR (423113) (Biolegend, USA), for 15 min in the dark at room temperature. Fluorescence minus-one (FMO) controls were used for gating as indicated in Supplementary Fig. S1. Cells were washed with 1 X PBS containing 1% cs-FCS then resuspended in 1 X Cell Fix solution (BD, USA) and analyzed using a LSRII flow cytometer (BD, USA) and FlowJo software version 10.1 (Treestar Inc., Ashland, Ore). Dead cells were excluded from the scatter plots prior to analysis and only single cellular populations were analyzed (Supplementary Fig. S1). Results were plotted as frequency (percentage of total), or expression as median fluorescence intensity (MFI) per number of double-positive cells.