Anti ly5.1 clone 6c3

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 incubated with anti‐CD45 (clone 30‐F11, BioLegend, San Diego, CA), anti‐EpCam (clone G8.8, BioLegend), anti‐Ly5.1 (clone 6C3, Miltenyi), anti‐Ulex Europeaus agglutinin I (clone FL‐1061, Vector Laboratories, Burlingame, CA), and anti‐Major Histocompatibility Complex (MHC) class II (clone M5/114.15.2, BioLegend) antibodies. Exclusion of dead cells was done adding 200 μg/ml of DAPI solution just before acquisition.
For the evaluation of epithelial stem‐progenitor markers, cells were incubated with anti‐EpCam (clone G8.8, BioLegend), anti‐OCT4 (clone EM92, Ebioscience), anti‐CD44 (clone IM7, BD, Biosciences, San Jose, CA), anti‐Sca‐1 (clone D7, BioLegend), anti‐CD90 (clone 30 H‐12, BioLegend), and anti‐MHC II (clone M5/114.15.2, BioLegend) antibodies.
At the time of sacrifice, mice were euthanized according to the ethical guidelines. Spleen, lymph nodes, and blood samples were prepared using standard protocols and analyzed with anti‐CD45 (clone 30‐F11, BioLegend), anti‐CD3 (clone 145‐2C11, BD, Biosciences), anti‐CD4 (clone RM4‐5, BD, Biosciences), and anti‐CD8 (clone 53–6.7, BD, Biosciences) antibodies.
Cells were acquired on a FACS‐Canto II or LSR Fortessa flow cytometer (BD, Biosciences) and analyses were performed using the FlowJo software (FlowJo, LLC, Ashland, OR).