Multiparameter Flow Cytometry Protocol

Cells were re-suspended in FACS buffer (PBS with 1% BSA, 2 mM EDTA) and incubated with Fc blocker for 10 min on ice. After washing with FACS buffer, cells were incubated with the desired antibodies at optimal concentrations for 20 min on ice in the dark. Cells were then washed with FACS buffer twice and re-suspended in 200 µL FACS buffer for flow cytometry analysis (LSRII, BD Biosciences, NJ, USA). For intracellular staining, cells were stained with surface markers as described above, treated with cell fixation/permeabilization kit reagents (BD Biosciences, NJ, USA) and then incubated with antibodies for desired intracellular markers. CD4-PE, CD4-APC, or CD4-eFluor 450 was purchased from BD Biosciences. Tfh cell staining was performed using a three-step staining protocol^{12 (link)}. Annexin V-pacific Blue (Biolegend, CA, USA) was used to assess the apoptotic status of cells. CellTrace Violet (CTV) (Thermo Fisher Scientific, MA, USA) was applied to identify cell proliferation status, and the proliferation index was calculated following the manufacturer’s instructions. Cell cycle status was assessed by 7-AAD (Tonbo Biosciences, CA, USA) and BrdU (Biolegend, CA, USA) staining. Flow cytometry analysis was performed using a LSRII platform (BD Biosciences, NJ, USA) and data were analyzed using FlowJo software 7.6 (TreeStar, CA, USA).