Anti cd4 clone sk3

Cells were washed once in MACS buffer (containing PBS, 1% BSA, 0.5 mM EDTA), centrifuged at 550 g for 5 min and re-suspended in 200 μL MACS buffer. Cells were stained in 100 μL of staining buffer containing anti-CD137, conjugated with phycoerythrin (PE, clone 4B4-1; BD Biosciences, USA), anti-CD8-PC5 (clone B9.11; Beckman Coulter 1:100), anti-CD4 (clone SK3; BioLegend, 1:200), anti-CD14 (clone 63D3; BioLegend, 1:200), and anti-CD19 (clone HIB19; BioLegend,1:200), all conjugated to Fluorescein isothiocyanate (FITC) for exclusion gates, for 30 min on ice. Samples were covered and incubated for 30 min on ice, washed twice in PBS, and resuspended in 1 mL PBS prior to analysis.

The titer of individual virus stocks was determined on SupT1 cells using the virus-encoded GFP signals measured by flow cytometry as readout for productive infection. Resting CD4 T cells were infected with virus stocks at different multiplicities of infection (MOIs) as indicated for each experiment. After 3 days, cells were washed twice with PBS and fixed with 4% PFA for 1.5 h. Cells were then washed and resuspended in FACS buffer. The percentage of GFP-positive cells was quantified by flow cytometry. Drug or antibody controls were added to cells 30 min prior to HIV-1 challenge. The following drugs were used: Efavirenz (EFV) (stock: 10 mM; Sigma Aldrich), AMD3100 (stock: 16 μg/mL; Sigma Aldrich), Anti-CD4 clone SK3 (stock: 25 μg/mL; Biolegend), T20 (stock: 90 mg/ml; Enfuvirtid; Roche), and Maraviroc (10 μg/μL; Sigma Aldrich).