Live dead l d fixable aqua stain

Flow cytometry measurements were performed using a CyAn ADP Analyzer (Beckman Coulter) and data were analyzed using version 9.8.2 of FlowJo software. Viable cells were detected by LIVE/DEAD (L/D) Fixable Aqua stain (Invitrogen) and the following antibodies were used for surface and intracellular staining: CD3e Pacific Blue, CD4 FITC, CD8 APC-Cy7, CD45.1 Pe-Cy7, CD25 PE, PD-1 PE, CTLA4 APC, IFNγ APC, FOXP3 PerCP-Cy5.5 (eBioscience). For staining of blood and splenocytes, cells were exposed for 5 min at RT to 0.155 NH₄Cl to lyse erythrocytes. The following staining steps were performed on ice. Cells were washed with PBS, stained for 15 min with Aqua L/D stain, resuspended in PBS + 2% FBS for surface staining for 15 min and finally fixed for 15 min in PBS + 2% PFA. IFNγ intracellular staining was performed in PBS + 2% FBS supplemented with 0.5% Saponin. Foxp3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent kit (ebioscience) was used for FOXP3 staining.