Mouse mTECs were isolated and purified as described previously45 pooling cells from 5-20 mice per experiment. The pre-enriched stromal cell fraction, sorted for unselected mature mTECs (n = 211 cells) was stained using the following antibodies: anti-CD45-PerCP (clone 30-F11, BD Pharmingen), anti-EpCAM-Alexa647 (G8.8, described by Farr et al.46 (link)), anti-CDR1-Pacific Blue (CDR1 hybridoma, described by Rouse et al.47 (link)) or anti-Ly51-FITC (clone 6C3, BD Biosciences), and anti-MHCII-PE (clone 16-10A1, BD Biosciences).
For the surface TRA-selected mTECs Tspan8 (n = 48) and Ceacam1 (n = 30), additional antibodies were added to the antibody mix namely: anti-I-A(b)-FITC (clone AF6-120.1, BD Pharmingen), anti-Tspan8-PE (clone 657909, R&D Systems) and anti-CD66a-PE (i.e., anti-Ceacam1, clone CC1, eBioscience). Dead cells were excluded using propidium iodide (PI) in a final concentration of 0.2 µg/ml. Cells were sorted on BD FACSAria™ III cell sorter (BD Biosciences) using the single-cell sorting mode as previously described10 (link). Single mature mTEC used in all the experiments represent cells from pooled thymic tissue.
For the surface TRA-selected mTECs Tspan8 (n = 48) and Ceacam1 (n = 30), additional antibodies were added to the antibody mix namely: anti-I-A(b)-FITC (clone AF6-120.1, BD Pharmingen), anti-Tspan8-PE (clone 657909, R&D Systems) and anti-CD66a-PE (i.e., anti-Ceacam1, clone CC1, eBioscience). Dead cells were excluded using propidium iodide (PI) in a final concentration of 0.2 µg/ml. Cells were sorted on BD FACSAria™ III cell sorter (BD Biosciences) using the single-cell sorting mode as previously described10 (link). Single mature mTEC used in all the experiments represent cells from pooled thymic tissue.