The following flow cytometry antibodies were obtained from BD Biosciences and Thermo Fisher Scientific: Anti-CD3 (SK7), anti-CD4 (RPA-T4), anti-CD8 (RPA-T8), anti-PD-1 (MIH4), anti-PD-L1 (MIH1), anti-TIGIT (MBSA43), anti-Ki67 (20Raj1), anti-CD38 (HIT2) and anti-HLA-DR (LN3). Live/Dead fixable Aqua (Life Technologies) was used for dead cell discrimination. For flow cytometry of EVs, 1μl aldehyde/sulphate latex beads (Thermo Fisher Scientific) were incubated with 4μl pEVs in 1ml double-filtered (0.2μm) PBS overnight at room temperature. The pEV-bead conjugates were blocked with glycine for 30mins, centrifuged at 5000 rpm for 5mins, washed twice with 1ml PBS supplemented with 0.5% Exo-free FBS and labelled with the following antibodies (BioLegend, anti-CD9 (HI9a), anti-CD63 (Ts63, Thermo Fisher), anti-CD81 (1D6-CD81, Thermo Fisher), anti-TSG101 (EPR7130B, abcam), anti-Flotillin1 (EPR6041, abcam), anti-TCRαβ (T10B9.1A-31, BD Biosciences), anti-HLA-G (BD Biosciences), anti-CCR5 (2D7, BD Biosciences), anti-CXCR4 (12G5, BD Biosciences), anti-PD-1 (MIH4, Thermo Fisher) and anti-PD-L1 (MIH1, Thermo Fisher). Intracellular cytokine staining for IFN-γ, T-bet, IL-17 and IL-4 was performed according to our previous reports (20 (link), 21 (link)). Data was acquired using a BD Fortessa flow cytometer followed by analysis using FlowJo v10.6 software.
Okoye I., Xu L., Oyegbami O., Shahbaz S., Pink D., Gao P., Sun X, & Elahi S. (2021). Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer. Frontiers in Immunology, 12, 697604.
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