Anti tcrβ pe

Blood samples were incubated with ACK lysis solution to remove erythrocytes. Single-cell suspension of splenic cells was obtained by gently pressing spleens through 70 μm cell strainers (Corning, cat. no. 352350). Bone marrow cells were harvested from one femur per mouse. Peritoneal lavage was performed by repeatedly flushing the peritoneal cavity with 10 ml PBS/2% FCS. Single-cell suspensions from the different tissues were resuspended in PBS/2% FCS and briefly incubated with Fc blocking antibodies (clone 2.4G2, purified in-house). Cells were then stained with the following fluorescently labelled monoclonal antibodies: anti-CD8α-FITC (ThermoFisher, cat. no. 11-0081-81), anti-TCRβ-PE (BioLegend, cat. no. 109208), anti-CD11b-PerCP-Cy5.5 (BioLegend, cat. no. 101228), anti-Gr-1-APC (ThermoFisher, cat. no. 17-5931-82), anti-CD19-PE-Cy7 (BioLegend, cat. no. 115520), anti-CD45-BV785 (BioLegend, cat. no. 103149), anti-CD4-BV711 (BioLegend, cat. no. 100447), anti-CD11c-BV605 (BioLegend, cat. no. 117333) and anti-Ly-6G-BV421 (BioLegend, cat. no. 127628). For nonviable cell exclusion, Fixable Viability Dye eFluor^® 780 (ThermoFisher, cat. no. 65-0865-18) was used. Stained cells were then fixed with 4% formalin and resuspended in PBS/2% FCS for flow cytometric analysis using a LSRFortessa (BD Biosciences). FACS data were then analysed with FlowJo software (RRID:SCR_008520, version 10.0.7).

Blood samples and splenocytes were treated with red blood cell lysis buffer and along with lymph node cells and thymocytes were washed with phosphate buffered saline (PBS). They were stained in FACS buffer (PBS, 10% FCS, 0.01% NaN₃) with anti-CD2 FITC, anti-TCRβ PE, anti-CD8 PerCP, anti-CD19 APC and anti-CD4 PB (all Biolegend). Live/Dead discrimination was performed by propidium iodide or Live Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies). Single cells were sorted (BD, MoFlow) onto AmpliGrid slides and processed immediately. For FACS analysis, cells were acquired at BD Canto II and Aria 5. Analysis was performed with FlowJo 9.4 and 10.