Isolation and Analysis of mTECs

mTECs were isolated, analyzed, and sorted as previously described (Michelson et al., 2022a (link)). Thymi were finely chopped, the lymphocyte-rich supernatant was removed, and the thymic pieces were incubated at 37°C in DMEM supplemented with 2% FCS, 25 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Lonza), 0.5 mg/ml collagenase (Sigma-Aldrich), and 0.1 mg/ml DNaseI (Sigma-Aldrich) for 15 min, then in the same medium with 0.5 mg/ml collagenase/dispase (Roche) for 15 min. To dissociate cell–cell interactions, 10 mM EDTA was added. To prepurify mTECs, cells were incubated with anti-CD45 microbeads (Miltenyi) for 15 min and then CD45⁺ cells were depleted using MACS LS columns (Miltenyi). Cells were then stained with antibodies against EpCAM, CD45, Ly51, A/E, Lypd8 (all Biolegend), and/or GP2 (MBL). DAPI (Sigma-Aldrich) and Fixable Yellow Live/Dead (Invitrogen) were used for dead cell exclusion. mTECs were defined as live CD45⁻ EpCAM⁺ Ly51⁻ cells and further gated as mTEC^hi or mTEC^lo based on MHCII levels. Flow cytometry was performed on LSRII or FACSymphony A1 instruments (BD), and cell sorting was performed on a FACSAria cell sorter (BD). Flow cytometry data were analyzed with Flowjo (BD).