Anti cd45 apc 30 f11

Thymi were excised from 5–6-wk-old age-matched mice, rinsed in PBS, cut into small pieces, collected in plain RPMI-1640, and incubated for 1 h at 4°C with gentle rotations to remove excess thymocytes. Supernatant was removed and remaining tissue pieces were incubated with Collagenase D (0.2 mg/ml; Roche), Dispase Grade I (0.2 mg/ml; Roche), and DNase-I (0.025 mg/ml; Roche) 2 times for 15 min at 37°C with mixing every 5 min. Cells were collected on ice and incubated with 5 mM EDTA for 10 min. Nonhematopoietic cells were enriched after staining with rat anti–mouse CD45 antibody (30-F11; eBioscience), followed by incubation with sheep anti–rat magnetic beads (Invitrogen) and magnetic depletion. Cells were stained before FACS sorting using the following antibodies: anti-CD45 APC (30-F11; eBioscience), anti-EpCAM eFluor450 (G8.8; eBioscience), anti-Ly51 FITC (6C3; BD), anti-CD31 FITC (390; eBioscience), anti-CD140a PE (APA5; eBioscience), UEA-1 Rhodamine (Vector Laboratories). Cells were sorted on a FACSAria II with 130 µm nozzle at low speed as follows: cTECs, CD45⁻EpCAM⁺Ly51⁺; mTECs, CD45⁻EpCAM⁺UEA-1⁺; endothelial cells, CD45^–CD31⁺; fibroblasts, CD45^–EpCAM^–CD31^–CD140a⁺. Sorted cells were of 94–96% purity, as determined by reanalysis.