Anti mouse cd19 pe cy7

Homogenized frozen spleen samples were sorted on an ARIA III instrument (BD, Franklin Lakes, NJ, USA) using the following antibodies: anti-mouse CD3 AF700, clone: 17A2, cat: 100216; anti-mouse CD19 PE/Cy7, clone: 6D5, cat. 115520; anti-mouse CD4 PE, clone: RM4-5, cat: 100,512 (Biolegend, San Diego, CA, USA); anti-mouse Vβ5.1/Vβ5.2 FITC, clone: MR9-4, cat: 51-01354L (BD, Franklin Lakes, NJ, USA); anti-mouse PD1 eFluor 450, clone: RMP1-30, cat: 48-9981-82; anti-mouse CD8 APC, clone: 53–6.7, cat: 17-0081-82 (eBiosciences, San Diego, CA, USA). Frozen OT1/OT2 splenocytes were thawed in RPMI medium (10% FCS, 1% pen/strep, 1% L-Glut) and filtered (30 µm, Sysmex, Goerlitz, Germany) before they were stained in sort buffer (PBS supplemented with 2% FCS and 0.025M HEPES) for 15 min at RT. Thereafter, cells were washed with sort buffer and sorted for CD4/CD8 Vb5pos/neg PD1pos/neg populations. Sorted T cell populations consisted of 1000 cells/population. Purity of sorted cell populations was checked regularly, and purity was found to be consistently high—T cell purity: 88.9–100%; CD4/CD8 purity: 88.6–99.7%; Vb5pos/neg PD1pos/neg populations: 97–100%. Different cell populations were sorted directly into RLT lysis buffer and RNA was immediately isolated after the sorting process using the RNeasy micro Kit (QIAGEN, Hilden, Germany).