Fig. 5

Detection of osteogenic differentiation of ASCs. ASCs harvested from subcutaneous (SC), retroperitoneal (RP), and lipoma (LP) fat sources were cultivated in the presence of osteogenic differentiation medium (ODM) for up to 21 days. a–d ASCs of SC, RP, and LP fat stained with Alizarin Red S (ARS) show osteogenic nodule formation and calcium deposition (red) in the mineralized matrix at day 21 post induction. Cells of the three groups were seeded in basal medium (BM). e Semi-quantification of alkaline phosphatase (ALP) activity as shown following osteogenic differentiation induction up to day 14 shows promoted ALP activity in all experimental groups including LP derived cells compared to those cells cultivated in BM condition. The p-nitrophenylphosphate is metabolized into p-nitrophenol (PNP) in the presence of ALP activity. The values of PNP induce a color change from colorless to yellow. f Semi-quantification of the bound Alizarin Red S (ARS) was measured by CPC at day 21 post osteogenic induction. Analysis of CPC absorbance at 562 nm revealed a higher osteogenic capacity of SC derived ASCs compared to RP and LP derived cells. g, h Expression fold change of ALP (f) and BMP2 (g) at days 7 and 14 post osteogenic differentiation induction shows upregulation of osteogenic markers in RP and SC derived cells compared to LP cells. The expression was normalized to non-induced cells in BM using 2^−∆∆cq method [30]. All data presented as mean ± SEM. *p < 0.05 and **p < 0.01. Scale bar = 100 μm