Anti human cd45 beads

For fluorescence-activated cell sorting (FACS), thymic stromal cells were enriched by Percoll-density gradient centrifugation (25 (link)) followed by depletion of CD45⁺ cells from the APC-enriched cell suspension (5x10⁸ cells) with anti-human CD45 beads (Miltenyi 130-045-801).subsequently stained with fluorochrome-conjugated antibodies. This stepwise enrichment lead to an approx. 200-fold proportional enrichment of TECs, fibroblasts and endothelial cells (Figures S2K, L). Propidium iodide was added to assess cell viability. 1-2x10⁵ live cells were sorted on a FACSAria fusion (BD Biosciences) and resuspended in RLT lysis buffer (Qiagen).

For the Legend Screen (BioLegend 700001), CD45⁺ cells were partially depleted from digested tissue (15x10⁸ cells) anti-human CD45 beads (Miltenyi 130-045-801) and mixed with 7x10⁷ undepleted cells. Cells were pre-mixed with antibodies against CD45 (BioLegend, 2D1) and EpCAM (BioLegend, 9C4).