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at 300500 mm from the growth cartilage and 150200 mm from the cortices. The cortical thickness was measured at midshaft diaphysis. The left femurs were fixed in 4% paraformaldehyde, decalcified with 5% EDTA for 1 week and embedded in paraffin. Five-mm thick sections were dewaxed and processed for TRAP staining by incubation for 30 mn at 37uC in substrate solutions containing naphthol AS B1 phosphate, sodium tartrate, fast red Methods Mice ERK12/2 mice, backcrossed on C57BL/6 background, and WT C57BL/6J mice were housed in a specific pathogen-free environment. All procedures were approved by the Animal Care Committee. Animal studies described herein were reviewed and approved ERK1 Regulates the Hematopoietic Stem Cell Niches violet and N,N dimetyl formamide in 20 ml acetate buffer. Sections were then rinsed under running tap water for 5 min, counterstained with fast green dye and mounted in coverquick. The surface of TRAP-stained cells was measured for the Oc.S/BS calculation using automatic image analyzer. Homing and lodging assays Total BM cells were isolated from donor animals. Cells were labeled with the cytoplasmic dye carboxyfluorescein diacetate succinimidyl diester according to manufacturer’s instructions. In homing assays, lethally irradiated C57BL/6 mice were injected with 156106 ERK12/2 or WT CFSEstained cells. Three and 24 hours after injection, the percentage of CFSE+ cells in BM was scored by flow cytometry. In lodging assays, 156106 CFSElabeled BM cells were transplanted into non irradiated mice. Three and 24 hours after injection, the percentage of CFSE+ cells in recipient-derived BM cells was scored by flow cytometry. The frequency of CFSE+ donor cells was determined as previously described. recombinant RANKL . The cultures were maintained for 5 days and re-fed every 2 days. Osteoclasts were identified by staining for TRAP activity, using the leukocyte acid phosphatase kit. TRAP-positive multinucleated cells with greater than three nuclei were counted as osteoclasts. RNA was isolated from the cells at day 5 of differentiation with cytokines to measure the expression of osteoclast-associated genes. To assess the osteoclast in vitro functionality, bone marrow-derived cells were differentiated in vitro for 4 days, and osteoclasts detached PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22179956 by treatment with 0,02% EDTA in PBS and numbered. For resorption test, the same number of osteoclasts from each set were lifted and seeded onto 24-well Osteo Assay dishes for an additional 24 h. OsteoAssay wells were subsequently stained with silver nitrate. The surface area was measured with ImageJ software, using an ImageJ plug-in developed by our imaging facility Deoxypyridinoline cross-link assays Bone related degradation products from type 1 collagen, deoxypyridinoline cross-links and creatinin were measured in evening urine using the Pyrilinks-D immunoassay and creatinin kit, according to the manufacturer’s protocols. Limiting dilution competitive bone marrow transplantation assay For each BMT experiment, 50, 100 or 250 LSK CD150+CD482 cells of each genotype were mixed with 256104 CD45.1/CD45.2 competitors, and subsequently transplanted into lethally irradiated recipient mice CD45.1. Peripheral blood in recipient mice was analyzed for the engraftment level of donor cells at 4 and 20 weeks after BM transplantation. Peripheral blood was obtained by Vercirnon manufacturer retro-orbital punction and blood leukocytes were obtained after hypotonic lysis. Multilineage hematopoietic engraftement was analyzed wit
