Sca1 brilliant violet 421

Cells were isolated from bone marrow, spleen, and thymus of mice, and stained with the following antibodies: B220 (APC; BD Pharmingen), cKit (PE-Cy7; BioLegend, San Diego, CA), CD3 (PE-Cy7; BD Bioscience), CD4 (APC-H7; BD Pharmingen), CD8 (ECD; Beckman Coulter), CD11b (PE; BD Bioscience), CD16/32 (PE; eBioscience, San Diego, CA), CD34 (FITC; BD Pharmingen), CD45.2 (PerCP-Cy5.5; BD Pharmingen), CD48 (APC-Cy7; BD Pharmingen), CD127 (ECD; BD Pharmingen), CD150 (PerCP-Cy5.5; BioLegend), Gr1 (APC-Alexa700; BD Bioscience), Lineage Cocktail (APC; BD Pharmingen), Sca1 (Brilliant Violet 421; BioLegend). Dead cells were excluded using either DAPI or Vivid-Aqua (Invitrogen) staining. All data acquisition was performed on a LSRII (BD) flow cytometer, and results were analyzed using FlowJo v.8.8.7 (TreeStar).

c-KIT-positive cells were enriched using a magnetic separation system (MACS) with anti-c-KIT magnetic beads (Miltenyi Biotec). The enriched cells were stained with c-KIT-APC (2B8, BioLegend), SCA-1-PE (E13–161.7, BioLegend), and lineage antibody cocktail (CD3, B220, CD11b, Gr-1, and Ter119; all from BD) conjugated with PE-Cy5. Dead cells were excluded using 7-amino-actinomycin-D staining. For the analysis of CXCR4 protein expression, c-KIT-enriched BM cells were stained with CXCR4-PE (2B11, eBioscience) or Isotype Control (eB149/10H5, eBioscience) together with CD48-FITC (HM48-1, BioLegend), CD150-PE-Cy7 (TC15-12F12.2, BioLegend), c-KIT-APC-eFluor780 (2B8, Fisher Scientific), SCA-1-Brilliant Violet 421 (D7, BioLegend), and lineage antibody cocktail conjugated with PE-Cy5. CD150⁺CD48⁻ within LSK cells were defined as HSCs (Kiel et al., 2005 (link)). Cells were sorted on FACSAria III or FACSAria IIu and analyzed on FACS Fortessa, LSRII, or FACSCanto II (BD). Collected data were analyzed with FlowJo software (Tree Star).