StemTAG™ Alkaline Phosphatase Staining and Activity Assay Kit, Colorimetric, 2 x 100 assaysUser Manual
|Research Area:||Cell Metabolism|
|Assay Category:||Kinase/ Phosphatase Assays|
|Detection Method:||ICC / Colorimetric|
- Analyze cell differentiation by monitoring active alkaline phosphatase (AP) using immunocytochemistry staining and a quantitative colorimetric activity assay
- Staining and activity assay protocols take less than 1 hour
- Suitable for human embryonic stem (ES) cells, embryonic germ (EG) cells, and embryonic carcinoma (EC) cells, as well as mouse ES and EG cells
Embryonic stem (ES) cells are continuous proliferating stem cell lines of embryonic origin first isolated from the inner cell mass (ICM). Two distinguishing features of ES cells are their ability to be maintained indefinitely in an undifferentiated state and their potential to develop into any cell within the body.
Based on previous methods developed for mouse ES cells, human ES cell lines were first established by Dr. James Thomson and colleagues. Like mouse ES cells, human ES cells express high levels of membrane alkaline phosphatase (AP) and Oct-4, a transcriptional factor critical to ICM and germline formation. However, unlike mouse ES cells, hES cells do not express stage-specific embryonic antigen (SSEA-1).
Although stem cells from different origins require different growth conditions for self-renewal and display different cell surface markers, AP is the most widely used stem cell marker.
AP staining of ES Cells. Murine embryonic stem cells (ES-D3) are maintained in an undifferentiated stage on gelatin-coated dishes in the presence of LIF, as indicated by the high AP activity. To induce differentiation, LIF was withdrawn over a period of several days; various differentiation events were observed (cells became flattened and enlarged with reduced proliferation). At the end of day 5, AP staining of differentiated cells was performed as described in the Staining Assay Protocol.
AP Activity Assay
pNP Standard Curve and AP Activity Assay. A: A serial 2-fold dilution of pNP standard was prepared in 1X Stop Solution, and the absorbance of each dilution was measured at 405 nm. B: Mouse embryonic D3 cells were grown in the presence or absence of LIF for 5 days. 10 ug of cell lysate was assayed for AP activity according to the Activity Assay Instructions.
The StemTAG™ Alkaline Phosphatase Complete Kit provides an efficient system for monitoring ES cell undifferentiation/differentiation through AP activity by both immunocytochemistry staining and quantitative activity assay.
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