Life Technologies
Alkaline Phosphatase (AP) Live Stain
A14353
Alkaline phosphatase (AP) is a well-known phenotypic marker of pluripotent stem cells such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). AP staining has been used as a marker which distinguishes pluripotent stem cells from feeder cells (e.g. mouse embryonic fibroblasts (MEF) and parental fibroblast cells) used in reprogramming experiments. However, currently available AP substrates are toxic to the cells and so the cells need to be fixed for staining, which prevents succession of cultivation after staining to check pluripotency.
To check pluripotency of ESCs and iPSCs, both mouse and human.
Human fibroblast cells were introduced with the 4 standard reprogramming factors (Oct4, Sox2, SSEA-4, and Tra1-60) and cultivated on feeder cells
1. Remove ESC/iPSC culture media. 2. Add 2mL of DMEM/F-12 (for 60mm dish) and aspirate for washing. 3. Dilute X500 AP live stain solution in DMEM/F-12 (4uL in 2mL of DMEM/F-12 for 60mm dish). 4. Add AP live stain solution to the cells. 5. Incubate cells at 37°C incubator for 20-30min. 6. Aspirate AP live stain solution and add 2 mL DMEM/F-12 for washing. 7. Repeat washing twice. 8. Visualize AP activity by using confocal/fluorescent microscope (FITC filter). 9. After removal of AP live stain solution from the media, fluorescent signal is lost within 2 hr and ESC/iPSCs can be continuously cultivated without loss of proliferation and pluripotency.
Wash thoroughly -- the washing steps should remove any excess substrate.
After several passages, the pluripotency of hiPSCs was checked by AP live stain. The hiPSCs stained positive (green color) for the pluripotent marker.
In general, AP is not a perfect marker for defining undifferentiated cells. Therefore, it is necessary to confirmed pluripotency by other surface markers, such as Oct4, Sox2, SSEA-1, Tra1-60 by immunostaining.
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