Alp apc

AD-MSC lines were washed with PBS and stained with ALP-APC (R&D) (1/50) and CD73-FITC (Biolegend) (1/160) for 25 min at 4 °C. Upon washing, the cell fractions (controls sorted, ALP+/CD73+, ALP−/CD73high, ALP−/CD73low) were sorted with a FACS BD Aria III 5L and seeded in Nunc™ 96-well plates (TPP) at a density of 1.2 × 10⁴ cells/cm² for osteogenic differentiation. Controls sorted were unstained cells processed through the FACS and collected without sorting specific subpopulations. Differentiation was induced 24 h after seeding. Freshly isolated SVFs were washed with PBS and stained with ALP-APC (R&D) (1/50), CD73-FITC (Biolegend) (1/160), and CD45-PE (Biolegend) (1/160) for 25 min at 4 °C. SVF fractions (controls sorted, CD45−/ALP+/CD73+, CD45−/ALP−/CD73high, CD45−/ALP−/CD73low) were sorted with FACS BD Aria III 5L and plated in vitro at a density of 1 × 10⁴ in 96-well plates (TPP) for osteogenic differentiation. All media were changed every 4 days.