Apc cy7 anti tcrβ

Cell suspensions were incubated with anti-mouse CD16/CD32 (Biolegend, 101,319). For cell surface flow cytometry, cells from spleens, thymuses and LNs were stained with specific antibodies for surface antigens as follows: PE-anti-CD4 (Biolegend, 100,408), Pacific Blue-anti-CD4 (Biolegend, 100,531), Brilliant Violet 510-anti-CD8a (Biolegend, 100,751), APC/Cy7-anti-TCRβ (Biolegend, 109,220), 7-AAD (BD Pharmingen™, 559,925), APC-anti-CD25 (Biolegend, 102,012), PerCP/Cy5.5-anti-CD44 (Biolegend, 103,032), PE/Cy7-anti-CD62L (Biolegend, 104,418), PE-anti-CD278 (ICOS) (Biolegend, 107,705), APC-anti-CD304 (Neuropilin-1, Nrp1) (Biolegend, 145,206), PE/Cy7-anti-CD279 (PD-1) (Biolegend, 109,110). For intracellular staining, cells were fixed and permeabilized with Fixation/Permeabilization Kit (eBioscience, 00–5123, 00–5223), washed with Permeabilization Buffer (eBioscience, 00–8333) and stained with PE-Cy7-anti-ki67 (eBioscience, 25-5698-82), PE-anti-CTLA4 (Biolegend, 106,306), AF488-anti-Foxp3 (Thermo Scientific™, 53-5773-82), PE-anti-Foxp3 (Biolegend, 126,404). For apoptosis analysis, cells were stained with FITC-AnnexinV (Biolegend, 640,906) in AnnexinV Binding Buffer (Biolegend, 422,201). Samples were analyzed by LSRII multicolor flow cytometer (BD Biosciences CA, USA) and data analysis was performed using the FlowJo software (Tree Star, USA).

2 × 10⁶ lymphocytes from spleens, thymuses and LNs were plated in round-bottomed 96-well plates in 1 ml RPMI complete media containing PMA (50 ng/ml, Sigma, P1585-1 MG), GolgiStop (1:1000, BD Biosciences, 554,724) and ionomycin (1 μM, CST, 9995S). After culturing for 5 h in 37 °C, 5% CO₂, cells were collected and stained with PE-anti-CD4 (Biolegend, 100,408), Brilliant Violet 510-anti-CD8a (Biolegend, 100,751), PerCP/Cy5.5-anti-CD44 (Biolegend, 103,032), APC/Cy7-anti-TCRβ (Biolegend, 109,220). Then cells were fixed, permeabilized and stained with APC-anti-IL-4 (Biolegend, 504,106), PE/CY7-anti–IFN–γ (Biolegend, 505,826) and Brilliant Violet 421-anti-IL-17A (Biolegend, 506,925).