Using cell-surface markers TNAP (tissue non-specific alkaline phosphatase) and c-KIT, we consistently obtained hPGCs of high purity, with >97% of cells from the unique TNAP-high and c-KIT-high population positive for alkaline phosphatase (AP) staining.
Cultures were stained for alkaline phosphatase (Sigma leukocyte kit) and colonies were scored as undifferentiated (alkaline phosphatase positive), differentiated (alkaline phosphatase negative) or mixed (partially alkaline phosphatase positive).
Cells were stained for alkaline phosphatase production using kit 86R (Sigma).
After several well-separated iPSC colonies were picked up for expansion, all the plates were stained for alkaline phosphatase (AP) using AP Staining Kit II (Stemgent, Lexington, MA, USA).
Sera were analyzed for alkaline phosphatase (ALP) using a commercial kit (Roche Diagnostics, Prague, Czech Republic).
The 5th passage GFP+ colonies were fixed in 4% paraformaldehyde (PFA) and stained for alkaline phosphatase (AP) activity using AP detection kit (Sigma Aldrich, Cat. No. 85L3R).
Differentiated samples were tested for alkaline phosphatase (ALP) activity by alkaline phosphatase kit (SIGMA-Aldrich) and calcium deposition by staining with 1% Alizarin red-S solution.
Cells were washed, fixed in 4% paraformaldehyde and tested for alkaline phosphatase (AP) activity using an AP kit (Millipore).
Osteogenic differentiation was checked with histological staining for alkaline phosphatase using the Alkaline Phosphatase, Leukocyte Kit 86-C (Sigma) and mineralized extracellular matrix was visualized by staining with alizarin red S (Sigma).
On days 1, 5, and 10, hMSC and cMSCs were stained for alkaline phosphatase (ALP) using a commercially available kit (Sigma-Aldrich).
