Thymocyte cell suspensions were stained for 20 min at room temperature with CD4-APC, CD8a-PE, TCRb-BV421 and CD69-FITC (BD-Pharmingen). Single cells were sorted into 96-well plates containing lysis buffer using a FACSAria Fusion flow cytometer (BD Biosciences) and the gates depicted in Supplementary Fig. 1 . Flow cytometry standard files were analysed with DIVA (BD Biosciences) and FlowJo v10 (TreeStar Inc) analysis software.
C57BL/6 (C57BL/6OlaHsd, Envigo, UK) and Mice lacking MHC class II expression41 (link) (JAX stock #003584) were maintained separately under specific pathogen-free conditions under Project Licences issued by the Home Office, UK. OT-I Rag2−/− and CD8.4 OT-I Rag2−/− mice32 (link) were maintained together at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, in accordance with laws of the Czech Republic. Six-week-old male or female mice were killed by cervical dislocation, and thymocyte or lymph-node cell suspensions were stained with CD4-APC, TCRb-FITC, CD69-BV421 (Pharmingen) or CD4-Alexa Fluor 700, CD8a-PE or CD8a-BV421, TCRb-APC (Biolegend), and LIVE/DEAD NIR (ThermoFisher). Data acquisition was on a Cytek Aurora flow cytometer (Cytek Biosciences) and flow cytometry standard files were analysed with FlowJo v10 (TreeStar Inc) analysis software. Data were further analysed using GraphPad Prism v5.04 (GraphPad Software).
C57BL/6 (C57BL/6OlaHsd, Envigo, UK) and Mice lacking MHC class II expression41 (link) (JAX stock #003584) were maintained separately under specific pathogen-free conditions under Project Licences issued by the Home Office, UK. OT-I Rag2−/− and CD8.4 OT-I Rag2−/− mice32 (link) were maintained together at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, in accordance with laws of the Czech Republic. Six-week-old male or female mice were killed by cervical dislocation, and thymocyte or lymph-node cell suspensions were stained with CD4-APC, TCRb-FITC, CD69-BV421 (Pharmingen) or CD4-Alexa Fluor 700, CD8a-PE or CD8a-BV421, TCRb-APC (Biolegend), and LIVE/DEAD NIR (ThermoFisher). Data acquisition was on a Cytek Aurora flow cytometer (Cytek Biosciences) and flow cytometry standard files were analysed with FlowJo v10 (TreeStar Inc) analysis software. Data were further analysed using GraphPad Prism v5.04 (GraphPad Software).
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