Multiparameter Flow Cytometry of Murine Spleen Cells

Spleen cells were resuspended in PBS-1% bovine serum albumin and incubated with anti-mouse CD16/CD32 (1:100, BD Fc Block) to block nonspecific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE, (1:400, 1:100, and 1:100, eBiosciences), T1ST2-FITC, (1:200, MD Biosciences), CD4-Brilliant, CD196 (CCR6)-PE (1:160, 1:640, BioLegend), CD25-PerCp-Cy5.5 (1:1,280, Thermo Fisher), and CD127-PE-Cy7 (1:320, Miltenyi Biotec, Germany). Viable cells were distinguished by means of a fixable viability dye APC-Cy7 (1:2,000, eBioscience). For detecting transcription factors, cells were first fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol and then stained with FoxP3-FITC (1:100, Thermo Fisher) and RorgT-APC (1:400, Biosciences). The specificity of all antibodies was assessed and optimal working dilutions were determined in a previously published study from our group (36 (link)).
Results were collected with BD FACS Canto II flow cytometer (Becton Dickinson) and analyzed with FlowLogic software (Inivai Technologies, Australia). The gating strategies are shown in Supplemental Fig. S2.