Murine TEC isolation was performed at the indicated time points following it-LV treatment. Murine thymus was cleaned of fat and stromal tissue, and then digested at 37°C with an enzymatic solution containing Liberase TL and DNAse I. Digested tissues were collected in DMEM with 10% fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin. Single thymic cell suspensions were then incubated with anti-CD45 micro-beads (Miltenyi Biotec) and processed with the AutoMACS Pro Separator (Miltenyi Biotec). The CD45 negative fraction was retrieved and then tested by multiparametric cytofluorimetric analysis for the expression of TEC markers.44 (link)
To check TEC enrichment after isolation, cells were immune-stained by anti-CD45 (clone 30-F11, BioLegend), anti-EpCAM (clone G8.8, BioLegend) and pan anti-major MHC class II (IA/IE MHC-II) (clone M5/114.15.2), anti-CD49F (GoH3) (BioLegend), anti-Ly5.1 (clone 6C3) (Miltenyi), and anti-CD80 (16-10A1) (BD Pharmingen). Exclusion of dead cells was done by adding 200 μg/mL of DAPI solution just before acquisition.
To check TEC enrichment after isolation, cells were immune-stained by anti-CD45 (clone 30-F11, BioLegend), anti-EpCAM (clone G8.8, BioLegend) and pan anti-major MHC class II (IA/IE MHC-II) (clone M5/114.15.2), anti-CD49F (GoH3) (BioLegend), anti-Ly5.1 (clone 6C3) (Miltenyi), and anti-CD80 (16-10A1) (BD Pharmingen). Exclusion of dead cells was done by adding 200 μg/mL of DAPI solution just before acquisition.