Multiparameter Flow Cytometry of Immune Cells

Splenocyte cell suspensions were resuspended in PBS blocking buffer containing 1% BSA and 5% FCS (Sigma-Aldrich) and incubated for 15 min at 4°C with Fc block CD16/CD32 antibodies (BD Biosciences; 5 μg/mL) to prevent non-antigen-specific binding. Cells (5 × 10⁵) were subsequently stained with antibodies (eBioscience, The Netherlands, unless otherwise stated) against CD69-APC, CD4-PerCP Cy5.5, CXCR3-PE, T1ST2-FITC, RORγ-APC, CD25-Alexa Fluor® 488, FoxP3-PE Cy7, CD196-PE, and Fixable Viability Dye-eFluor® 780 (eBioscience, USA) or matching isotype controls for 30 min at 4°C. Cells were fixed using fixation buffer (eBioscience) or permeabilized for intracellular staining using the intracellular staining buffer set (eBioscience) according to the manufacturer's protocol. Flow cytometry was performed using FACS Canto II (BD Biosciences), and results were analyzed using Flowjo Software V. 10.6.2 (Becton Dickinson & Company (BD). We used fluorescence minus one (FMO) to differentiate between negative and positive staining cell populations (3 (link), 22 (link)).