Anti p24 fitc clone kc57

To assess cell surface expression of proteins, flow cytometry was performed. Cells were washed with PBS and subsequently stained with the viability dye LIVE/DEAD Fixable Near-IR (Life Technologies) and with the antibodies anti-CD3-BUV395 (clone UCHT1, BD), anti-CD4-BV711 (clone RPA-T4, Biolegend), anti-CD155-PE (clone SKIL4, Biolegend), anti-HLA-ABC-Pe-Cy7 (clone W6/32, Biolegend), anti-HLA-E-BV421 (clone 3D12, Biolegend), anti-tetherin-APC (clone RS38E, Biolegend) and anti-IgG1-PE isotype control (clone MOPC21, Biolegend) for 15 min at RT. After washing the cells with PBS, an intracellular staining was performed. In brief, cells were incubated in BD Cytofix/Cytoperm for 20 min at 4°C, washed with BD Perm/Wash buffer and stained with anti-p24-FITC (clone KC57, Beckman Coulter) for 20 min. After another washing step, cells were fixed in BD Cellfix and analyzed by flow cytometry (BD LSR Fortessa). HIV-1-infected cells were defined as p24 Gag⁺ CD4^dim and uninfected as p24 Gag^- CD4⁺ cells. Cells infected with HIV-1 ΔNef mutant viruses were defined as p24 Gag⁺ and tetherin^- cell, as Nef-deficient viruses are not able to downregulate CD4.