Analysis of in vivo cell proliferation by BrdU or EdU incorporation

Cell proliferation was determined using a BrdU labeling kit (BD bioscience) or a Click-iT™ Plus EdU Flow Cytometry Assay Kits (Invitrogen). Twenty four hours before sacrifice, BrdU was administrated by intraperitoneal injection (2 mg/mouse in 200 µl PBS) as a single dose. At indicated time points, LSK, GMP, CMP, MEP cells were sorted from BM BMMC. Sorted cells were fixed in 70% ethanol overnight at –20°C, denatured in 2N HCl/0.5% Triton X-100 for 20 minutes at room temperature, neutralized with 0.1 M sodium borate for 5 minutes, and stained with anti-BrdU antibody (BD Biosciences, MD USA) for 30 minutes at room temperature in PBS/0.5% BSA/0.5% Tween 20. Cells were then resuspended in 500 µl PBS containing 10 µg RNase A and 5 µg Propidium Iodide (PI), incubated for 30 minutes, and immediately analyzed by flow cytometry. The mean frequencies of BrdU+ cells in the HSC and each progenitor populations were measured. For the EdU incorporation assay, EdU was administrated by intraperitoneal injection (0.5 mg/mouse in 200 µl PBS) as a single dose 24 hr before sacrificing the mice. The isolated cells were prepared and stained following a protocol provided by the manufacturer ((Invitrogen).