The aspirated blood was collected into a 1.5 mL tube per mouse. Then, the femur and tibia from the left limb of each mouse were dissected and transferred to a 15 mL conical tube containing Hanks’ balanced salt solution supplemented with 1% fetal bovine serum (FBS). PBMCs and BM-KSL cells, which are stem cell populations of EPCs, were isolated as previously reported [29 (link)].
Lineage-negative BM cells (BM-Lin-) were incubated with saturating concentrations of directly labeled anti-c-Kit (dilution, 1:25; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Sca-1 antibodies (at 1:25 dilution) (BD Biosciences) for 30 min on ice. Afterward, BM-KSL cells were isolated by live sterile cell sorting using a BD FACSAria™ Fusion flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Lineage-negative BM cells (BM-Lin-) were incubated with saturating concentrations of directly labeled anti-c-Kit (dilution, 1:25; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Sca-1 antibodies (at 1:25 dilution) (BD Biosciences) for 30 min on ice. Afterward, BM-KSL cells were isolated by live sterile cell sorting using a BD FACSAria™ Fusion flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Free full text: Click here
