Fbs imdm

Bone marrow cells were collected by aspiration and 5 × 10⁶ cells were stained with PE-Flt3, APC-Sca-1, APC-Cy7-c-kit, and PerCP-lineage antibodies. The cells were incubated with 5 μM DCF-DA (Sigma) in 10% FBS IMDM (Gibco) at 37°C for 30 min and then analyzed by FACSCantoll (BD Bioscience) (52).

Single-cell suspensions prepared from BM, peripheral blood (PB), and spleen were incubated for 30 min at 4 °C with fluorochrome–antibodies (BioLegend). Dead cells were excluded by FVD eFluor® 455UV (eBioscience) staining. Flow cytometry was performed using CytoFLEX (Beckman), LSR Fortessa, or FACSAria II (BD) flow cytometer. Antibodies used in this study are available in Supplementary Methods.
For cell cycle or quiescence analysis of HSCs, the BM cells were incubated with fluorochrome–antibodies, fixed and permeabilized with Transcription Factor Buffer Set (BD), and stained with 5 μg/ml DAPI (Sigma) or PE-anti-Ki67 (BioLegend).
For Akt phosphorylation assay, live Lin⁻ BM cells were sorted, starved in 2% FBS/IMDM (Gibco) for 1 h, and then treated with 5 ng/ml GM-CSF (Peprotech) at 37 °C for indicated time. After stimulation, the cells were fixed, permeabilized, and stained with APC-Cy7-anti-CD117, PerCP-Cy5.5-anti-Sca1, anti-p-Akt (Cell Signaling), and PE-anti-IgG (eBioscience).