Leukocyte alkaline phosphatase kit

After osteogenic induction for 7d, cells in 6-well plates were stained by Leukocyte Alkaline Phosphatase Kits (Sigma-Aldrich, USA) for ALP staining. And Alizarin Red S staining was performed to detect the mineralized nodule formation after BMSCs were incubated with osteogenic medium for 21d. All the staining was performed according to the manufacturer’s instructions.

Cells were fixed in 4% paraformaldehyde for 10 min at 4°C and incubated overnight at 4°C with anti-Nanog antibodies. After a brief wash with phosphate-buffered saline + 0.1% Tween-20, the cells were detected in parallel by Cy3 fluorescence emissions, respectively. Nuclei were stained with Hoechst 33342 (Molecular Probes). The images were captured using a BIOREVO BZ-9000 fluorescence microscope (Keyence). Alkaline phosphatase assays to evaluate the pluripotent state were performed using leukocyte Alkaline Phosphatase kits (Sigma-Aldrich).

The cells cultured in 24-well plates were fixed with 4% paraformaldehyde (Solarbio, Beijing, China), and their alkaline phosphatase activity was measured to assess stem cell identity [85 (link)]. This was performed by Leukocyte Alkaline Phosphatase Kits (Sigma); the procedure for staining went according to the manufacturer’s protocols.

Alkaline phosphatase staining was carried out with the leukocyte alkaline phosphatase kit (Sigma, #86R) according to the manufacturer’s protocol.

Mineralization was induced on confluent monolayers in 12-well plates by addition of osteogenic media. Monolayers were washed with 1X PBS and fixed for 1 h with cold ethanol (70%, v/v), then washed 3 times with excess dH₂O prior to addition of 1 mL of Alizarin Red (2% w/v, pH 4.2) per well. The plates were incubated in the dark at RT for 10 min. After removal of unincorporated dye, wells were washed three times with dH₂O, re-aspirated, and stored at room temperature (RT). In identical cultures for the mineralization assay, monolayers of MPC1 and MPC2 cells were washed twice with 1X PBS and fixed for 30 min in 4% PFA. PFA was removed and cells were washed again in 1X PBS, then stained using the Leukocyte Alkaline Phosphatase Kit (Sigma) as previously described^{18 (link)}. Plates were stored at RT. Images were taken at 10X magnification on an inverted Leica microscope. Alizarin red staining was quantified using cetylpyridinium chloride (CPC) extraction for 3 h as previously described^{19 (link)} and absorbance was measured at 544 nm. Intensity of alkaline phosphatase staining was quantified using ImageJ, as previously described^{20 (link)}.