Intravascular Labeling of Human Cells

Human CD45⁺ hematopoietic cells in the blood and the organ vasculature of HSPC-engrafted MISTRG mice were labeled as previously described (31 (link)). For this purpose, 2 μg of a phycoerythrin (PE)-conjugated anti-human CD45 antibody (Biolegend, clone HI30) was injected intravenously (IV) 5 minutes before organ harvest without any prior perfusion. After isolation from various organs (bone marrow, spleen, liver, lung), cells were stained ex vivo with an APC-Cy7-conjugated anti-human CD45 antibody and other cell surface antibodies to determine the frequency of intravascular (IV CD45-PE⁺) and extravascular (IV CD45-PE^-) ILCs by flow cytometry. Blood was also analyzed by flow cytometry to confirm successful intravascular labelling, defined as >90% IV CD45-PE⁺ human cells in blood.