Biotin anti lineage cocktail

Antibodies were from Pharmingen unless otherwise specified. Peripheral blood was obtained by lancing the facial vein and collecting several drops into EDTA microtainers (Pharmingen). Blood counts were obtained using a Hemavet 50FS. Blood elements were enumerated using PE or PerCP-Cy5.5-anti-CD3 (145-2C11), PE-anti-B220 (RA3-6B2), APC-anti-CD19 (1D3), APC, PE or PerCP-Cy5.5-anti-CD11b (M1/70), PE or PerCP-Cy5.5-anti-Ter119 (Ter119), and PerCP-Cy5.5 or APC-anti-Gr-1 (RB6-8C5, Biolegend). Stem and progenitor cells were enumerated using biotin-anti-Lineage Cocktail, PerCP-Cy5.5-streptavidin, APC-anti-c-Kit (2B8) and PE-Cy7-anti-Sca-1 (D7, eBioscience), in addition to PE-anti-CD16/CD32 (FcγR, 2.4G2) and Brilliant Violet 421-anti-CD34 (RAM34) for CMP, GMP, and MEP, PE-Texas Red-anti-CD127 (IL7R, SB199) for CLP, or Brilliant Violet 421-anti-CD34 and PE-anti-CD135 (FLT3, A2F10.1) for MPP, ST-HSC, and LT-HSC. Alternatively, LSK cells were stained using PE—anti-CD150 (Q38-480) and Brilliant Violet 421–anti-CD48 (HM48-1) for LSK/SLAM LT-HSC. Marrow subsets for RNA analysis were obtained after lineage-depletion and antibody staining via a FACSAria II cell sorter (BD Biosciences).

C57BL/6 Vav-NUP98-HOXD13 and Cebpa +37 kb Enh/Prom-hCD4 transgenic mice were previously described [16 (link), 17 ]. Marrow was obtained by flushing femurs and tibias with phosphate-buffered saline. GMP, CMP, and Lin^-Sca-1⁺c-Kit⁺ (LSK) marrow cells were enumerated, after red blood cell lysis with ammonium chloride, using biotin-anti-Lineage Cocktail, PerCP-Cy5.5-streptavidin, APC-anti-c-Kit (2B8), PE-Cy7-anti-Sca-1 (D7, eBioscience), PE-anti-CD16/CD32 (FcγR, 2.4G2), and Brilliant Violet 421-anti-CD34 (RAM34). Human CD4 was detected using FITC-anti-hCD4 (RPA-T4). Antibodies were from Pharmingen unless otherwise specified. Marrow subsets for RNA analysis were obtained after lineage-depletion, using biotin-conjugated B220, Gr-1, CD11b, Ter119, and CD3 mouse Lineage Cocktail (BD Pharmingen), anti-biotin microbeads, MACS columns (Miltenyi Biotec), and antibody staining via a FACS Aria II cell sorter (BD Biosciences).