Cd90 2 53 2

To prepare single cell suspension, ventral and dorsal sheets of the ears were separated from the cartilage and incubated for 90 min in CO2 incubator at 37°C in 1 mL volume of RPMI 1640 (Sigma-Aldrich, #R7388) containing 0.25 mg ml⁻¹ Liberase TL (Roche Diagnostics, #5401020001). The digested ears were passed through a 3 mL syringe to make single-cell suspension. The cells were filtered through 70 μm nylon mesh and washed in FACS buffer at 1500 rpm for 5 minutes. Cells were suspended in FACS buffer for further analysis. For surface staining the following antibodies were used at 1:100 dilutions in FACS buffer according to the manufacture’s specifications. CD45 (30-F11, eBiosciences), CD3 (17A2, eBiosciences), CD90.2 (53–2.1, eBiosciences), βTCR (H57–597, eBiosciences), CD4 (RM4–5, Biolegend), CD8 (YTS5167.7, eBiosciences), CD11b (M1/70, eBiosciences), Ly6G (1A8, eBiosciences), and Ly6C (AL-21, BD Pharmingen). For counting the cells AccuCount Fluorecent particles (Spherotech) were used. The stained cells were run on BD FACSymphony^™A3 (BD Biosciences) and the acquired data were analyzed using FlowJo software (Tree Star).

Murine Fc receptors were blocked before staining using supernatant from hybridoma 2.4G2 (rat anti-CD16/32). Monoclonal antibodies (mAbs) were diluted in FACS Buffer (1% BSA, 0.01% NaN3, PBS) to final concentrations that were empirically determined by pilot staining experiments. The following mAbs clones were used: CD11b (M1/70, eBioscience, 1:400), CD11c (N418, Biolegend, 1:200), Ly6C (Hk1.4, eBioscience, 1:200), Ly6G (1A8, BioLegend, 1:200), CD90.2 (53–2.1, eBioscience, 1:200), IgM (11/41, eBioscience, 1:200), CD64 (X54/5/7.1, BioLegend, 1:200), CD80 (16–10A1, eBioscience, 1:200), MHCII (I-A/I-E) (M5/114.15.2, eBioscience, 1:300), F480 (CL-A3–1, BioRad, 1:200), IFNGR1/CD119 (2E2, BD Bioscience, 1:100), ICAM-1 (CD54) (YN1/1.7.4,eBioscience, 1:100). IFNGR1 (CD119) was stained using a secondary Streptavidin-APC (eBioscience). Cells were analyzed on a BD LSR Fortessa (BD Biosciences) and data were processed with FlowJo software (Treestar).