Anti sca 1 antibodies

BM-MSC phenotypes were analyzed by flow cytometry using a FACSCalibur (BD Biosciences, NJ, USA). A total of 1 × 10⁶ cells/mL were incubated with FITC-, PerCP-, PE-, or APC-conjugated anti-CD34, anti-CD45, anti-CD29, anti-CD44, anti-CD90, anti-c-kit, or anti-Sca-1 antibodies (BD Biosciences) in phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) for 30 minutes at room temperature.
Adipogenic differentiation was induced by culture with adipogenic medium supplemented with 10⁻⁸M dexamethasone and 10⁻⁴M L-ascorbic-acid-2-phosphate (Sigma-Aldrich, St. Louis, MO) as previously described (Ngoc et al., 2011) [32 (link)]. After 30 days of induction, cells were fixed in 3% formaldehyde in PBS for 10 minutes and stained with Oil Red O.
Osteogenic differentiation was induced by incubation with culture medium that consisted of ascorbic acid, dexamethasone, 6-glycerol phosphate (Sigma-Aldrich, St. Louis, MO), and calcium deposits which were visualized by alizarin red.

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).