Anti cd31 microbead

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′.

PBMCs from adult leukocyte reduction filters [21 (link),22 (link)], CB samples, and peripheral blood were isolated by Ficoll^™ gradient centrifugation (Biochrom, Berlin, Germany). CD4⁺CD45RA⁺CD31⁺ naive T cells were enriched, using a CD4⁺ T cell Isolation Kit II and AutoMACS (magnetic-assisted cell sorting) (Miltenyi Biotec, Bergisch-Gladbach, Germany) followed by negative selection by CD45RO Micro Beads and positive selection by anti-CD31 Micro Beads (Miltenyi Biotec) The purified CD45RA⁺ fractions contained 95–97% CD45RA⁺ cells, and the purified CD31⁺ fractions contained 87–99% CD31⁺ T cells. The cells were cultivated at 37°C in RPMI 1640 medium (Biochrom, Berlin, Germany), supplemented with penicillin-streptomycin and with 10% human AB-plasma. A total of 2×10⁶ T cells/ml were loaded with anti-CD3 Ab (0.5 μg/ml) plus anti-CD28 Ab (0.5 μg/ml) for two minutes, then stimulation was performed by cross-linking the Abs with 10 μg/ml goat anti-mouse IgG (GAMIg) (Invitrogen, Darmstadt, Germany).

Primary mouse brain microvessel endothelial cells were isolated according to the reported methodology^{39 (link)}. Briefly, 8-week-old healthy C57/BL6 WT mice were sacrificed and brain tissue was isolated. The tissue sample was fragmented into small pieces and digested with Liberase Blendzyme (0.625 mg/ml, Roche) at 37 °C on a rotator for 1 h. The brain cells were then seeded in Collagen type I coated flasks in EndoPM culture medium (1% Endothelial Cell Growth Supplement (ECGS), 1% antibiotic solution, and 5% fetal bovine serum in Endothelial Cell Medium). The cellular suspension was maintained in the incubator for 14 days. Endothelial cells were then enriched with magnetic sorting with anti-CD31 microbeads (Miltenyi) with auto-MACS (Miltenyi) and expanded before subjected to further experiments.