Multiparametric Flow Cytometry Analysis

Single-cell suspensions from spleens, lymph nodes, and thymi were incubated with anti-CD16/32 at 5 µg/ml for 20 min on ice to block Fc receptors. Cells were stained with the following antibodies: CD4 PerCP-Cy55, TCRβ PerCp-Cy55, and CD62L APC (TONBO); CD44 FITC, PD-1 PerCP-Cy55, CD69 Cy7PE, CD25 PerCP-Cy55 (Biolegend); CD4 BUV395, and CD8 BUV737 (BD); Vα2 PE (phycoerythrin), Vα2 Pacific Blue, Vα2 FITC, FR4 Cy7PE, and CD73 BV605 (eBioscience); and CD25 BV605 (Life Technology). Dead cells were excluded using the live/dead fixable Near-IR death cell stain kit (Invitrogen). Intracellular Foxp3-FITC staining was done according to the manufacturer’s instructions (Life Technology). For detection of negatively selected thymocytes, caspase 3 PE (BD) was used as previously described (Breed et al., 2019 (link)). For intracellular flow cytometry, antibodies against phosphorylated ERK^T202/Y204 (mAb 197G2), AKT^S473 (Cell Signaling) were used as previously described (Hsu et al., 2009 (link)). Antibody against Alexa 647–anti-rabbit IgG was used as a secondary antibody to detect phosphorylation of ERK and AKT.