Biotin conjugated anti mouse lineage antibodies

Hematopoietic stem cells and uncommitted progenitors (LKS +: Lin − Kit + Sca-1 +) and committed progenitors (LKS −: Lin − Kit + Sca-1 − CD16/32 +/− CD34 +/− representing CMP: common myeloid progenitors, GMP: granulocytic-macrophage progenitors and MEP: myelo-erythroid progenitors) were isolated by staining with a biotinylated cocktail to label and eliminate mature cells (anti-CD5, anti-CD11b, anti-B220, anti-7-4, anti-Gr1, anti-Ter119, Miltenyi Biotec), followed by eFluor 450-streptavidin, APC-anti-c-Kit, PE-anti-Sca-1, PE-Cy7-anti-CD16/32 and Alexa-Fluor 700-anti-CD34 to label HSPC. LKS + sub-populations were labeled using biotin-conjugated anti-mouse lineage antibodies (Miltenyi Biotec) followed by PE-Texas Red-streptavidin (eBioscience), APC-anti-c-Kit, PECy7-anti-Sca-1, FITC-anti-CD34 and PE-anti-Flk2 (LT-HSC: LKS + CD34 − Flk2 −, ST-HSC: LKS + CD34 + Flk2 − and MPP: LKS + CD34 + Flk2 +). Then cell sorting was performed on a BD Influx cell sorter.

Hematopoietic Stem and Progenitors Cells (HSPC) were labeled using biotin-conjugated anti-mouse lineage antibodies (Miltenyi Biotec) followed by eFluor 450- streptavidin (eBioscience), APC-anti-c-Kit, PE-anti-Sca-1, PE-Cy7-anti-CD16/32 and Alexa-Fluor 700-anti-CD34. Mature cells were identified as follows: granulocytes were labeled with APC-anti-Gr1 and PE-anti-CD11b; myeloid cells were positive for CD11b and/or Gr1; lymphoid cells were identified with PECy7-anti-B220 for B lymphocytes, APC-anti-CD3 for T lymphocytes, PE-anti-CD49b and anti-PE-Cy7-anti-NKG2D for Natural Killer (NK) cells. Cells were washed then data were acquired either on a LSRII (Becton Dickinson) or on a CyAn ADP (Beckman Coulter) and analyzed using FlowJo software (Treestar).