Flow Cytometry

For intravenous (IV) labeling, mice were injected intravenous with 2 μg of anti-CD45 or anti-CD8 antibody 3 minutes before euthanizing mice. Single-cell suspensions were stained with antibodies in FACS buffer (1%BSA in PBS) with brilliant staining buffer for 30 minutes at 4C. Antibodies were purchased from Biolegend, Cell Signaling Technologies, Invitrogen, BD Biosciences, and Tonbo and included, CD8 (53–6.7), CD62L (MEL-14), CD45.1 (A20), CD90.1 (OX-7), CD90.2 (30-H12), CXCR6 (SA051D1), CD69 (H1.2F3), CD103 (2E7), CD44 (IM7), CD45 (30-F11), and TCF1 (C63D9). Ghost dye 780 (ZombieRed) was used to detect dying cells. For tetramer staining, single cell suspensions were incubated with H2-K^b-B8R_20–27 and H2-D^b-GP_33–41 tetramers for 45 minutes at room temperature in the dark. Tetramers were provided by the NIH Tetramer Core. Staining of intracellular TCF1 was performed with True-Nuclear Transcription Buffer set as per manufacturer suggestions. Samples were analyzed with a BD FACSymphony and Flowjo software.