Once mice were euthanized, the animals were immediately passed through an air lock chamber and then into a temperature-, humidity-, O2-, and CO2- controlled, custom-built glove box (Hypoxic Chamber, Coy Lab Products) (23). The glove box was maintained at 3% O2 by N2 balance and 5% CO2. Femurs were collected and flushed in sterile PBS (HyClone, GE Healthcare Life Sciences) within the glove box. Collected BM cells were then split in half, with one half kept in the hypoxic glove box and the other half moved to a biosafety hood exposed to ambient air conditions (~21% O2), where they remained for 1 hour prior to further processing so that the BM cells could acclimate to ambient air conditions, as we described previously (23). Subsequent procedures such as staining cells for flow cytometry or setting up colony assays were performed simultaneously in the hypoxic glove box or in the biosafety cabinet under ambient air conditions (see Figure 1A for the experimental design). When culturing cells under hypoxic conditions, the cells were equilibrated in the hypoxic glove box, such that at no point were the cells exposed to ambient air conditions, and the cells were then transferred from the glove box to an incubator maintained at 5% O2 by N2 balance and 5% CO2 via airtight containers. All solutions (e.g., fixatives), media, reagents (e.g., antibodies), plasticware, pipette tips, sterile instruments, and gauze, as well as anything else that could come into contact with the femurs and cells (including the 70% ethanol used to sterilize the mouse prior to removal of the femur) used for collection and processing under hypoxia were pre-equilibrated to hypoxic conditions in the 3% O2 glove box for at least 18 hours prior to use. All liquids (especially the methylcellulose used for colony assays because of its viscosity) were vortexed vigorously to displace as much oxygenated air as possible. We emphasize the absolute requirement for extensive equilibration of all materials to the hypoxic conditions of the glove box prior to use and rigorous attention to details to obtain stable and reproducible results for the hypoxia-collected/processed cells. The reagents used for ambient air–acclimated cells were kept under ambient air conditions.

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