Fitc clone 30 f11

4T1 tumors were resected, minced, and digested in Liberase TL (Roche, Basel, Switzerland) and DNAse (Roche, Basel, Switzerland) for 30 min at 37 °C under constant rotation. Single cell suspension was filtered through 70 μm strainers and washed with PBS. Cells were then incubated with Live Dead NIR in PBS for 20 min and then Fc blocking antibody (2.4G2, Biolegend, San Diego, CA, USA) in FACS buffer at 4 °C for 15 min. Fluorochrome-conjugated antibodies specific to cell surface markers were added and incubated for 45 min at 4 °C. Fluorochrome-conjugated antibodies to CD45 (1:500 FITC clone 30-F11), CD3 (1:100 BV785 clone 17A2), CD8 (1:300 APC clone 53-6.7), CD4 (1:300 BB700 clone RM4-5), MHC-II (1:200 BV605 clone M5/114.15.2), and CD11c (1:200 AF700 clone N418) were obtained from BioLegend (San Diego, CA, USA). The stained samples were acquired in an Aurora cytometer (Cytek, Fremont, CA, USA) and data was analyzed using FlowJo version 10.8 (FlowJo LLC, Ashland, OR, USA).

The femurs, tibiae, iliac crest, humeri, and vertebrae of 6-16 weeks old mice were crushed, washed with 10 mL of PBS supplemented with 2% heat-inactivated FBS, and strained through 70-μm meshes. Cell suspension was depleted of red blood cells by ammonium chloride lysis (STEMCELL Technologies), and stained with the lineage depletion kit (19816A, STEMCELL Technologies) following the manufacturer’s instructions and passed through magnetic columns. Lineage-depleted cells were resuspended in 100 μl of PBS supplemented with 2% FCS containing the following antibodies against: c-Kit (APC-Cy7, clone 2B8, 105826, Biolegend), Sca-1 (BV421, clone D7, 108128, Biolegend), CD45 (FITC, clone 30-F11, 103108, Biolegend), Flt3 (PE, clone A2F10, 135306, Biolegend) and Il-7R⍺ (BV605, clone A7R34, 135041, Biolegend). Cells were incubated at 4°C for 30 minutes in the dark, washed, and resuspended in 100 μl of PBS supplemented with 2% FCS containing streptavidin (BV510, 405234, Biolegend). Cells were further incubated at 4°C for 15 minutes, washed and resuspended in 500 μl of PBS supplemented with 2% FCS containing 0.5 μl 7AAD (A1310, Life Technologies). Cells (lineage^- c-Kit⁺ population, and lineage^- c-Kit^lo Sca-1⁺ population) were bulk sorted using a Becton Dickinson Influx sorter.