BrdU incorporation in vivo was measured by flow-cytometry using the APC BrdU Flow Kit (BD Biosciences, San Jose, CA). Mice were given an intraperitoneal injection of 1 mg of BrdU (Sigma, St. Louis, MO, St. Louis, MO) per 6 g of body mass in PBS and maintained on 1 mg/ml of BrdU in the drinking water for 24 hours. Cell cycle analysis in vitro was performed as follows. 500 HSCs were sorted into SF-O3 medium containing SCF and TPO (see below) and cultured for 3 days. BrdU (10 μM final concentration) was added for an hour before cells were cytospun to a slide. Slides were fixed with cold methanol for 5 minutes at −20 °C, then washed with PBS containing 0.01 % NP-40 and treated with 2N HCl for 20 minutes. Slides were blocked in PBS containing 4 % goat serum, 4 mg/ml BSA and 0.1% NP-40 followed by staining overnight at 4 °C with antibodies against BrdU (BU1/75, 1:100, Abcam, Cambridge, MA) and phospho-Histone H3 Serine10 (3H10, 1:2500, Millipore, Temecula, CA) diluted in blocking buffer. Primary antibody staining was developed with secondary antibodies conjugated to Alexa fluor 488 or 594 (Invitrogen, Eugene, OR) together with DAPI (2 μg/ml). Slides were analyzed on an Olympus microscope equipped with 40× objective lens.
For Ki-67/propidium iodide staining, HSCs were sorted into 70% ethanol and kept at - 20°C for at least 24 hours. Ki-67 staining was performed using a FITC Ki-67 kit (BD Biosciences), followed by staining with 50μg/ml propidium iodide (Molecular Probes, Eugene, OR) and analyzed by flow-cytometry.
For Ki-67/propidium iodide staining, HSCs were sorted into 70% ethanol and kept at - 20°C for at least 24 hours. Ki-67 staining was performed using a FITC Ki-67 kit (BD Biosciences), followed by staining with 50μg/ml propidium iodide (Molecular Probes, Eugene, OR) and analyzed by flow-cytometry.
