C kit apc eflour780 2b8

Multiparameter FACS analyses were performed to determine populations of HSCs (Lineage⁻Sca-1⁺c-Kit⁺Flk2⁻CD48⁻CD150⁺), multipotent progenitor sub-population 1 (MPP1, Lineage⁻Sca-1⁺c-Kit⁺Flk2⁻CD48⁻CD150⁻), MPP2 (Lineage⁻Sca-1⁺c-Kit⁺Flk2⁻CD48⁺CD150⁺), and MPP3 (Lineage⁻Sca-1⁺c-Kit⁺Flk2⁻CD48⁺CD150⁻). Antibodies were purchased from eBiosciences, San Diego, CA, unless otherwise noted. BM cells freshly harvested from femurs and tibias were stained with biotin-labeled antibodies against mouse hematopoietic lineage markers: Mac-1 (M1/70), Gr-1 (RB6-8C5), Ter119 (TER-119), CD3e (145-2C11), B220 (RA3-6B2), and subsequently stained with antibodies conjugated with various fluorochromes: streptavidin eFluor® 450, Sca-1-PE-Cy7 (D7, BD Biosciences, San Jose, CA), c-Kit-APC-eFlour780 (2B8), CD150-Alexa Fluor® 647 (TC15-12FF12.2, BD Biosciences), CD48-FITC (HM48-1), Flk2-PE (A2F10.1, BD Biosciences) for HSCs or MPPs. Specific cell populations were gated based on immune phenotypes for quantification or cell sorting. Fluorescence Minus One (FMO) was used for setting the gating on control samples. Flow cytometric analyses were performed on BD LSR II (BD Biosciences, San Jose). Cell sorting was conducted using BD FACSAria. Data were analyzed using FlowJo software (TreeStar, Ashland).