Cd4 apc rpa t4

For flow cytometric analysis of BLaER1 transdifferentiation and surface marker expression, 1 × 10⁶ native or transdifferentiated BLaER1 cells were collected, washed once in FACS buffer (10% FCS, 0.1% Sodium acetate in PBS; 10 min, 300g, 4°C) and stained with CD11b-APC (M1/70, Biolegend), CD19-PE (HIB19, Biolegend), CD14-PacBlue (M5E2, Biolegend), CD163-PE (GHI/61, BD), CD206-APC (19.2, BD), CD11c-VioBlue (MJ4–27G12, Miltenyi), CD4-APC (RPA-T4, Biolegend), CXCR4-PE (12G5, BD), CCR5-PE (T21/8, Biolegend) or respective isotype controls (Biolegend, BD, Miltenyi) and Fixable Viability Dye eFluor 780 (Thermo Fischer) in presence of FC Block (BD, 20 min, 4°C). Stained cells were washed in FACS buffer twice and fixed in 2% paraformaldehyde (30 min, RT), before analyzing on a LSR II instrument (BD). For readout of HIV-1-mCherry infection, up to six wells of a 96-well plate were pooled and stained with CD11b-APC and Fixable Viability Dye eFluor 780 as detailed above. For intracellular SAMHD1 staining, cells were fixed in Cytofix Buffer (BD; 37°C, 10 min), subsequent to cell surface staining, and permeabilisation with Perm Buffer III (BD; 2min on ice), before staining with anti-SAMHD1 (12586–1-AP, Proteintech) or an isotype control (CST; 60 min, RT) and anti-rabbit IgG-DyLight 405 (Thermo Fischer; 60 min RT). Infected cells were analyzed on a BD LSRFortessa.