Bone marrow was harvested from femur, tibia, and hip bones in mice. Total bone marrow was depleted of differentiated hematopoietic cells (lineage-positive cells) using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD). Magnetically sorted Lineage-negative (lin−) cells were kept overnight (O/N) at 4°C in IMDM medium supplemented with 10% FBS (HyClone) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Staining was performed for 20 min at room temperature (RT) using CD3ε (Lin)–APC clone 145-2C11 (553066; BD), Ter-119 (Lin)–APC clone Ter-119 (557909; BD), CD45R/B220 (Lin)–APC clone RA3-6B2 (553092; BD), Ly6G-6C (Lin)–APC clone RB6-8C5 (553129; BD), Ly-6A/E (Sca-1)–PeCy7 clone d7 (558162; BD), CD117 (c-Kit)–PE or PerCP-Cy5.5, clone 2B8 (553355 or 560557, respectively; BD), CD34–FITC or AF700 clone RAM34 (560238 or 560518; BD), CD135 (Flk2)–BV421 or PE clone A2F10.1 (562898 or 553842, respectively; BD). HSCs (Lin−Sca+c-Kit+CD34lowFlk2−) were sorted using ARIA3, ARIA Fusion, or Influx cell sorters (BD) and collected in Stem Span (StemCell).
When the cells were irradiated in vitro, HSCs were cultured in medium containing Flt3-Ligand, IL-3, IL-6, SCF as described (de Laval et al., 2013 (link)) in the presence or absence of TNF-α. TNF-α was added to the medium at 1 µg/ml 1 h before IR.
When the cells were irradiated in vitro, HSCs were cultured in medium containing Flt3-Ligand, IL-3, IL-6, SCF as described (de Laval et al., 2013 (link)) in the presence or absence of TNF-α. TNF-α was added to the medium at 1 µg/ml 1 h before IR.