Cd3ε clone 145 2c11

Total bone marrow (BM) cells were isolated from mouse femurs and tibias by grinding the tissues in RPMI 1640 medium (WelGENE, Gyeongsan-si, South Korea) containing 2% fetal bovine serum (FBS). Red blood cells (RBCs) were lysed in the presence or absence of ACK buffer (150 mM NH₄Cl, 1.0 mM KHCO₃ and 0.1 mM EDTA [pH 7.4]), and filtered through a strainer. Antibodies were purchased from BD Biosciences or BioLegend. BM cells were stained as previously described [31 (link)] and analysed using FACSCanto II (BD Biosciences, Qume Drive San Jose, CA, USA). For peripheral blood analysis, cells were washed with PBS, and RBCs were lysed using RBC lysis buffer (BioLegend, San Diego, CA, USA). Fc receptors were blocked with an unlabelled CD16/32 antibody (clone 93; BioLegend, San Diego, CA, USA). The cells were washed, and extracellular marker proteins were stained for 30 min at 4 °C with fluorophore-conjugated antibodies specific for CD45 (clone 30-F11), CD11b (clone M1/70), Ly6G (clone 1A8), Ly6C (clone AL-21) and CD3ε (clone 145-2C11) from BD Pharmingen, and CD45R/B220 (clone RA3-6B2) from BioLegend. The cells were washed twice and analysed using a Gallios™ flow cytometer (Beckman Coulter, CA, USA). Data were analysed using the FlowJo software (Biosciences, Franklin Lakes, NJ, USA).

Lymphocytes were counted and subjected to viability staining (Fixable Aqua Dead Cell staining, Life Technologies, #L34957) and subsequently to receptor Fc blockade (BD #553142). Staining was performed using the following antibodies: CD4 (clone RM4-4, BD), CD45 (clone 30-F11 eBioscience), CD45.1 (clone A20, BD), CD45.2 (clone 104, BD) CD8a (clone 53-6.7, eBioscience or BD), CD8b (clone YTS156.7.7, BioLegend), CD11b (clone RM2817, Thermo Fisher), Ly6c (clone AL-21, BD), CD3ε (clone 145-2C11, BD), CD19 (clone 1D3, BD), CD90.2 (53-2.1, BD), CD127 (clone A7R34, BD), CX3CR1 (clone SA011F11, BioLegend), CXCR3 (clone CXCR3-173, Biolegend), CD103 (clone 2E7, eBioscience or Biolegend), CD69 (clone H1.2F3, BD), TCR-β (clone H57-597, BD, TCR-γδ (clone eBio-GL3, eBioscience). H2-K^b:SIINFEKL or H2-M3:fMIGWII tetramers were either produced in house or, for the former, obtained thorugh the NIH tetramer core. Samples were fixed (IC Fixation Buffer, eBioscience), washed, resuspended in FACS buffer and acquired with a LSRII flow cytometer (BD) either immediately or on the following day. For intracellular staining, the following antibodies were used: IFN-ϒ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) in Permeabilization buffer (eBioscience).