Bone marrow endothelial cells were isolated in accordance with the published protocol by Poulos et al. (16 (link)). Intravital labeling of ECs was achieved via intravenous injection of 25 μg of anti–VE-cadherin AF-647 (BioLegend®, San Diego, CA) into wild-type C57BL/6 mice. Mice were euthanized 15 min after injection. Femurs and tibias were dissected, cleaned of adventitia, and crushed with a mortar and pestle. Crushed BM was digested for 10 min at 37°C in 2.5 mg/ml Collagenase A and 1 unit/ml Dispase II (Life Technologies, Carlsbad, CA). Cells were rinsed in ice-cold phosphate buffered saline (PBS) with 1% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Waltham, MA) and depleted of lineage-positive cells with the Miltenyi lineage depletion kit (Miltenyi Biotec Inc., San Diego, CA). The lineage-negative fraction was stained with anti-CD45-V450 and 7AAD (both from BD Biosciences). Cells were sorted on a BD FACSAria™ Fusion (model no. 656700-10-H-3201, BD Biosciences). BM ECs were isolated via staining for the 7AAD−CD45− VE-cadherin+ fraction. Cells from five mice were pooled together to comprise one biological replicate.