Cd4 bv785 gk1.5

Lungs were digested with 0.52 U/ml Liberase TM (Roche) and 60 U/ml DNase I (Roche) in RPMI 1640 (Cellgro), at 37°C for 30 min. Lung leukocyte enrichment was performed by using a 30% Percoll gradient and the cell suspension obtained after erythrocyte lysis. Single cells were then incubated with anti-mouse CD16/32 (93, TruStain fcX, BioLegend) to block Fc receptors. Staining was performed with Fixable Viability Dye eF780 (eBioscience), to identify dead cells, and the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD45-PerCP/Cy5.5 (30-F11), CD4-BV785 (GK1.5) (all from BioLegend), and CD3e-BUV395 (145-2C11) (from BD Biosciences). At least 1,000,000 events were recorded for each sample. Only viable and non-doublet cells were considered. Flow cytometric analysis was performed using a LSRFortessa X-20 flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

Cells isolated from mouse spleen and thymus were stained with Ghost Violet™ 510 viability dye (TONBO Biosciences) for dead cell exclusion, blocked with Fc block (2.4G2, BD Biosciences) and stained with the following anti-mouse antibodies from Biolegend: CD4-BV785 (GK1.5), CD44-PE-Cy7 (IM7), TCRb-APC (H57-597), CD8-PerCP-Cy5.5 (53-6.7) and CD45-APC-Cy7 (30-F11). Cells were then fixed and permeabilized using fixation and intracellular staining permeabilization wash buffer from Biolegend following the manufacturer’s instructions. Cells were stained with mouse anti-IRAP-AF594 (F5, Santa Cruz Biotechnology, dilution 1/20) or with rabbit a-Stx6 (ProteinTech, dilution 1/100) or monoclonal rabbit IgG (Cell Signaling Technology, dilution 1/500) followed by staining with a-rabbit AF594 (Thermo Fisher Scientific, dilution 1/200).