Pe cy7 conjugated sca 1

BM collection, LSK staining, and sorting were done as described before[19 (link)]. Briefly, BM was made from either male or female 6 week-old C57BL/6 WT mice. Long bones from all four limbs of each mouse were flushed thoroughly using 27G1/2 needle with 8-10 mL of heparin medium consisting of Hank’s Balanced Salt Solution (HBSS) supplemented with 1 % Penicillin/Streptomycin, and 20 U/mL heparin. Flushed BM cells were layered on top of Ficoll (hydrophilic polysaccharide) and centrifuged at 1500 rpm for 30 min at room temperature. Low-density BM cells were collected at the interface of medium and Ficoll. These low-density BM cells were washed once with stain wash (PBS, 1% bovine calf serum, and 1% penicillin-streptomycin) followed by antibody staining for 15 min on ice. The following cocktail of antibodies was used: phycoerythrin (PE)-conjugated CD3, CD4, CD45R, Ter119, and Gr1; AF700-conjugated c-Kit (CD117, eBioscience); PE-Cy7-conjugated Sca-1; fluorescein isothiocyanate (FITC)-conjugated CD34; allophycocyanin (APC)-conjugated Flk2 (eBioscience). Unless noted otherwise, all monoclonal antibodies were obtained from BD Biosciences. Cells were washed once more with stain wash buffer, and data was acquired using a BD LSRII (BD Biosciences).