Lsr 2 flow cytometry system

The adherent NPC cells were digested it into single cell suspension and then fixed with BD Cytofix™ buffer (Cat. No. 554655) for 20 minutes at room temperature. The cells were permeabilized with BD Phosflow™ Perm Buffer I (Cat. No. 557885), and then stained with antibody Nestin (561231, Becton, Dickinson and Company;), Sox2 (ab75485, Cambridge, USA), Vimentin (ab128507, Cambridge, USA), Tuj1(ab195879, Cambridge, USA), Map2 (560399, Becton, Dickinson and Company), GFAP (ab4674, Cambridge, USA) and theire Isotype control (550795, Becton, Dickinson and Company; ab170190, Cambridge, USA; ab91356, Cambridge, USA; ab171464 Cambridge, USA; 557721, Becton, Dickinson and Company; ab37382, Cambridge, USA). Flow cytometry was performed on a BD LSR™ II flow cytometry system (Becton-Dickinson, San Jose, CA) and the data were analyzed with BD FACSDiva Software v6.1.3.

Non-activated PBMC were infected or not with the indicated HIV strains for 2 hours at 37°C. Free virions were then washed with PBS. The cells were resuspended in fresh media and incubated 5 hours at 37°C in the presence of PE/CY7 anti-CD107a antibody (clone H4A3 Biolegend #328617) and in the presence of GolgiStop™ (BD Biosciences #554724) and GolgiPlug™ (BD Biosciences #555029) for intracellular cytokine staining. The cells were then harvested and stained for viability (Fixable blue viability dye, Life Technologies #L-23105) for 30 min, then stained with PE-CD3 (clone HIT3a Biolegend #300307), V500-CD8 (clone RPA-T8 BD horizon #560775) and BV605-CD4 (clone OKT4 Biolegend #317437). After fixation and permeabilization (BD Cytofix/Cytoperm™ #554722) the cells were stained for Pacific blue-Perforin (clone B-D48 Biolegend #353305), APC-IFN-γ (clone B27 Biolegend #506510), FITC-TNF-α (clone MAb11 BD PharMingen #554512), PerCP-Cy5.5-MIP1-β (clone D21-13551 RUO BD PharMingen #560688) and AlexaFluor^® 700-GranzymeB (clone GB11 BD PharMingen #561016). Fluorescence signals were then quantified using a BD LSR-II flow cytometry system (Becton Dickinson) with FACSDiva8 software and results were analyzed with FlowJo software.