Brilliant violet 421 conjugated anti cd34 clone ram34

Miltenyi AutoMacs CD117-enriched bone marrow cells were stained for 1 h at 20°C protected from light with a mix of biotin-conjugated antibodies (including anti-CD3e (clone 145–2C11, BioLegend), anti-CD4 (clone RM4–5, Thermo Fisher), anti-CD8 (clone 53–6.7), anti-CD19 (clone 6.D5, BioLegend), anti-CD127 (clone B12–1, BD), anti-B220 (clone RA3–6B2, BioLegend), and anti-Ter119 (clone TER-119, BioLegend)), as well as PE-Cy7-conjugated anti-SCA-1 (clone D7, BD), Brilliant Violet 785-conjugated anti-LY6C (clone HK1.4, BioLegend), PerCp Cy5.5-conjugated anti-LY6G (clone 1A8, BioLegend), Brilliant Violet 421-conjugated anti-CD34 (clone RAM34, BD), APC-conjugated anti-CD117 (clone 2B8, BioLegend), and Brilliant Violet 605-conjugated anti-CD115 (clone T38–320, BD), PE- Cy5 conjugated CD135 and PerCP-eFluor710-conjugated anti-CD16/CD32 (clone 93, Thermo Fisher). Cells were then washed twice with FACS buffer and incubated for 15 min at 20°C with streptavidin-APC-Cy7 (BD). Cells were washed once in FACS buffer, and resuspended in FACS buffer for analyses.

AutoMacs CD117-enriched BM cells were stained for 1 h at 20°C protected from light with a mix of biotin-conjugated antibodies (including anti-CD3e (clone 145–2C11, BioLegend), anti-CD4 (clone RM4–5, Thermo Fisher), anti-CD8 (clone 53–6.7), anti-CD19 (clone 6.D5, BioLegend), anti-CD127 (clone B12–1, BD), anti-B220 (clone RA3–6B2, BioLegend), and anti-Ter119 (clone TER-119, BioLegend)), together with PE-Cy7-conjugated anti-SCA-1 (clone D7, BD), Brilliant Violet 785-conjugated anti-LY6C (clone HK1.4, BioLegend), PerCp Cy5.5-conjugated anti-LY6G (clone 1A8, BioLegend), Brilliant Violet 421-conjugated anti-CD34 (clone RAM34, BD), APC-conjugated anti-CD117 (clone 2B8, BioLegend) and Brilliant Violet 605-conjugated anti-CD115 (clone T38–320, BD), PE- Cy5 conjugated CD135 and BUV395-conjugated anti-CD16/CD32 (clone 93, Thermo Fisher). MDPs (Lin-, KIT+, SCA-, CD16/32-, CD34hi, CD135+, CD115+ ), GMP(Lin-, KIT+, SCA-, CD16/32+,CD34hi) and C-GMP (Lin-, KIT+, SCA-, CD16/32+/−,CD34hi) (Fig. 3A), were sorted using FACSAria II (Becton, Dickenson, and Company) and cells were collected in a solution of DPBS + 50% FBS (Atlanta Biologicals). Other granulocytic and monocytic lineage progenitors stages were sorted based on the gating strategy shown in Extended data Fig.1E.