Isolation and Analysis of Thymic Stromal Cells

Digested thymuses (34 (link)) were depleted of CD45⁺ cells using anti-CD45 microbeads (Miltenyi Biotec), in conjunction with LD columns. Cells were stained with Abs to CD45 (30-F11), EpCAM1 (G8.8), TER-119 (TER-119), and podoplanin (8.1.1), and CD45⁻EpCAM1⁺ thymic epithelial cells (TEC) and CD45⁻EpCAM1⁻podoplanin⁺ mesenchymes were FACS sorted using a MoFlo XDP (Beckman Coulter). CD31⁺ endothelial cells were sorted using anti-CD31 microbeads (Miltenyi Biotec) and MS columns, according to the manufacturer’s instructions. Sorted populations were analyzed by quantitative PCR (qPCR) for expression of the indicated genes exactly as described (36 (link)). Primer sequences are as follows: Actb (NM_007393) QuantiTect Mm_Actb_1_SG primer assay (Qiagen QT00095242); Ccl19 NM_(011888.2), forward, 5′-GCTAATGATGCGGAAGACTG-3′, reverse, 5′-ACTCACATCGACTCTCTAGG-3′; Ccl21a (NM_011124.4), forward, 5′-ATCCCGGCAATCCTGTTCTC-3′, reverse, 5′-GGGGCTTTGTTTCCCTGGG-3′; Ccl25 (NM_009138.3), forward, 5′-TTACCAGCACAGGATCAAATGG-3′, reverse, 5′-CGGAAGTAGAATCTCACAGCA-3′; Cxcl12 (NM_021704.3), forward, 5′-GCTCTGCATCAGTGACGGTA-3′, reverse, 5′-TGTCTGTTGTTGTTCTTCAGC-3′; Kitl (NM_013598.2), forward, 5′-CCCTGAAGACTCGGGCCTA-3′, reverse, 5′-CAATTACAAGCGAAATGAGAGCC-3′; Selp (NM011347.2), forward, 5′-CATCTGGTTCAGTGCTTTGATCT-3′, reverse, 5′-ACCCGTGAGTTATTCCATGAGT-3′.