Cd69 h1.2f3 pe cy7

Approximately 2 million splenocytes, 2 million lung-derived lymphocytes, 4 million BM cells, and lysed whole blood were used for dextramer staining. Cells were stained using the commercial H-2 Db ASNENMETM-APC and H-2 Db SSLENFRAYV-PE (Immudex, Virum, Denmark) for 20 min at RT in the dark. Surface staining was added containing the following antibodies: CD44(IM7)-BrilliantBlue515, CD103(2E7)-BrilliantBlue700, CD4(RM4-5)-AF700, Fixable Viability Stain 780, CD8a(53-6.7)-BrilliantViolet510, CD62L(MEL-14)-BrilliantViolet650, CD49a(Ha31/8)-BrilliantViolet786, CD69(H1.2F3)-PE/Cy7, KLRG-1(2F1)-PE-CF594, CD3e(145-2C11)-Brilliant UV395 (all BD Biosciences), and CD127-A7R34-BV711 (Biolegend). These were incubated for 30 min at 4 degrees. After washing twice, cells were resuspended in FACS buffer. Acquisition was performed on an LSR Fortessa X-20 (BD), and data analyses were performed using FlowJo v10.6.2 (BD).

Around 2 million lymphocytes were stimulated with ASN, SSL, or SIIN peptide for 6 h. Monensin (Biolegend) was added to the cells for the last 5 h, followed by storage o/n at 4 °C. The next day, cells were washed twice with FACS buffer (2 mM EDTA, 0.5% BSA in PBS) and extracellular staining was performed for 30 min at 4 °C in 100 µL FACS buffer with the following antibody mix: CD44(IM7)-BrilliantBlue515, CD103(2E7)-BrilliantBlue700, CD4(RM4-5)-AF700, Fixable Viability Stain 780, CD8a(53-6.7)-BrilliantViolet510, CD62L(MEL-14)-BrilliantViolet650, CD49a(Ha31/8)-BrilliantViolet786, CD69(H1.2F3)-PE/Cy7, KLRG-1(2F1)-PE-CF594, CD3e(145-2C11)-Brilliant UV395 (all BD Biosciences), and CD127-(A7R34)-BV711 (Biolegend). After washing, cells were fixated and permeabilized with BD Cytofix/Cytopmerm kit (BD Biosciences) according to the manufacturer’s protocol. Cells were then stained intracellularly with IFNγ(XMG1.2)-APC, IL-2(RMP1-30)-BrilliantViolet421 and TNF(MP6-XT22)-PE (all BD) for 20 min at 4 °C. After washing twice, each pellet was resuspended in FACS buffer and measured on an LSR Fortessa X-20 (BD). Data were analyzed using FlowJoTM Software v10.6.2 (BD). IFNγ responses were not corrected for medium background responses.