Multiparametric Flow Cytometry Analysis

Single-cell suspensions were prepared from spleens or from cell cultures. Antibodies for surface staining, PerCP-Cy5.5-conjugated anti-CD3, PE-conjugated anti-CD4, FITC-conjugated anti-PD-1, APC-conjugated anti-CXCR5, Alexa Fluor 647-conjugated anti-GL7 and FITC-conjugated anti-PD-1 were purchased from BD bioscience. For intracellular staining, splenocytes were first stained with surface markers followed by fixation and permeabilization using FoxP3 Staining Buffer set purchased from eBioscience53 (link). Cells were labelled intracellularly PE-conjugated anti-Bcl-6 (eBioscience, San Diego, CA, USA), PE-conjugated anti-IL-17, APC- conjugated anti-IFN-γ or PerCP-Cy5.5- conjugated anti-FoxP3 (BD Biosciences, San Diego, CA, USA). Flow cytometry was performed on 4-laser/13-color BD LSRFortessa (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, Ashland, OR).