Sca 1 percp cy5

Bone marrow was harvested from the femurs, tibias, and pelvic bones of mice by crushing. c-Kit⁺ cells were enriched using anti-c-Kit microbeads (Cat #130-091-224, Miltenyi Biotech, San Diego, CA) followed by magnetic separation (autoMACS Pro Separator; Miltenyi Biotech). Previously enriched c-Kit⁺ cells were incubated with a cocktail of biotin-conjugated monoclonal antibodies against CD5, CD11b, B220, 7-4, Gr-1, and Ter-119 (Cat. No. 130-090-858, Miltenyi Biotech). Enriched c-Kit⁺ cells were stained with antibodies to define HSPC populations as follows^{42 (link)}: HSC [Streptavidin-BV605 (Biolegend), Sca-1-PerCP-Cy5.5 (E13-161.7) (Biolegend), c-Kit-APC-780 (2B8) (Thermo Fisher Scientific, Waltham, MA), CD48-APC (HM48-1) (Biolegend), CD150-PE-Cy7 (TC15-12F12.2) (Biolegend)]; MPPs/CLPs [Streptavidin-BV605, Sca-1-PerCP-Cy5.5, c-Kit-APC-780, CD135-APC (A2510) (Biolegend), CD127-PECy7 (A7R34) (Tonbo Biosciences)]; CMPs/GMPs/MEPs (Streptavidin-BV605, Sca-1-PerCP-Cy5.5, c-Kit-APC-780, CD32/16-PECy7 (93) (Biolegend), CD34-A647(RAM34) (BD Biosciences)]. Dilutions used and catalogue identifiers for each antibody are provided in Supplementary Table 2. 4’,6-diamidino-2-phenylindole (DAPI) was used to exclude dead cells. Cells were analyzed on a FACSAria III (BD Biosciences, San Diego, CA) and FlowJo20 version 9.4.11 (Tree Star, Ashland, OR) was used to analyse related data.

Cardiotoxin (CTX) from Naja mossambica mossambica was purchased from Sigma Aldrich (St. Louis, MO, USA). For immunohistochemical staining, Pax7, bromodeoxyuridine (BrdU) and myosin heavy chain (MF20) antibodies were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Ly6b.2 (clone 7/4) and CD11b were purchased from AbD Serotec (Raleigh, NC). For fluorescence-activated cell sorting (FACS), propidium iodide (PI), CD31-APC, CD45-APC, Sca1-PerCP-Cy5.5, Biotinylated-Vcam1; PE/Cy7-streptavidin, Ly6G/C-PE-Cy7, F4/80-Alexa Fluor 488, and CD206-PE were purchased from BioLegend (San Diego, CA). Calcein Blue Viability Dye was purchased from eBioscience (San Diego, CA).