Magnetic bead conjugated anti cd45 antibody

For the analysis of TECs, minced thymuses were digested with 1 unit per ml Liberase (Roche) in the presence of 0.01% DNase I (Roche). Single-cell suspensions were stained for the expression of CD326 (EpCAM, BioLegend), CD45 (eBioscience), CD249 (Ly51, eBioscience), H-2K^b (BioLegend) and I-A^b (BioLegend), and for the reactivity with UEA-1 (Vector Laboratories). For the intracellular staining of β5t, surface-stained cells were fixed in 2% (g/vol) paraformaldehyde, permeabilized in 0.05% saponin, and stained with rabbit anti-β5t antibody followed by AlexaFluor488-conjugated anti-rabbit IgG antibody. For the isolation of TECs, CD45⁻ cells were enriched with magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). For the analysis of thymocytes and splenocytes, cells were stained for the expression of CD4, CD8 and TCRβ (BioLegend). Multicolor flow cytometry and cell sorting were performed on FACSAriaII (BD Biosciences).

For the analysis of thymic epithelial cells (TECs), minced thymuses were digested with 0.5 unit/ml Liberase (Roche) in the presence of 0.02% DNase I (Roche). Single-cell suspensions were stained with antibodies specific for EpCAM (BioLegend, Cat# 118216, RRID:AB_1236471), CD45 (eBioscience, Cat# 48045180, RRID:AB_1518807), Ly51 (BioLegend, Cat# 108308, RRID:AB_313365, Cat# 108312, RRID:AB_2099613), Podoplanin (BioLegend, Cat# 127418, RRID:AB_2629804), I-Ab (BioLegend, Cat# 116406, RRID:AB_313725), and for the reactivity with UEA-1 (Vector Laboratories, Cat# B-1065, RRID:AB_2336766, Cat# FL-1061, RRID:AB_2336767, Cat# DL-1068). For the analysis of Aire and DCLK1, surface-stained cells were fixed in 5% formaldehyde neutral buffer solution (Nacarai Tesque), permeabilized in 1× permeabilization buffer (eBioscience), and stained with anti-Aire (eBioscience, Cat# 50593482, RRID:AB_2574257) or anti-DCLK1 (abcam, Cat# ab31704, RRID:AB_873537) antibody. For the isolation of TECs, CD45− cells were enriched in magnetic bead conjugated anti-CD45 antibody (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse, FACSAria II, and LSRFortessa (BD Biosciences).

Minced thymuses were digested with 0.5 unit/mL Liberase TM (Roche) in the presence of 0.02% DNase I (Roche). CD45⁻ cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec). CD45⁺ cell-depleted single-cell suspensions were stained for the expression of EpCAM (CD326, BioLegend, clone G8.8), CD45 (BioLegend, clone 30-F11), Ly51 (CD249, BioLegend, clone 6C3), and for the reactivity with UEA1 (Vector Laboratories). Cell sorting of cTECs were performed on FACSAria II (BD Biosciences).