Alkaline phosphatase (AP) is highly expressed in pluripotent stem cells, includingembryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the iPSCsinducing process, the activation of AP is also necessary for successful reprogramming.In traditional AP detection methods, cells are fixed and cannot be used for furtherstudies. This kit provides a fast, simple and sensitive method for AP detection withoutfixing the cells. The reagent is nontoxic to the cells and the fluorescent signal will bedischarged from the cells by exocytosis completely within two hours after removal ofthe staining solution, allowing the cell being used for following studies.

ProcedureThe amount of reagents used in this protocol is base on a 24-well plate, for otherplates and dishes, please make the corresponding adjustments.

1. Remove the growth medium from the cultures.

2. Wash with 500μL of 37°C DMEM/F12 for 3 minutes, repeat two times.(DMEM/F12 should be pre heated to 37°C before use)

3. Dilute the 500x AP Live Solution directly to the cells and incubate in a 37°C CO2incubator for 30 minutes.

4. Remove the working solution, wash with 500μL DMEM/F12 for 3 minutes, repeattwo times.

5. Add 500μL DMEM/F12 to the cells and observe the result under a fluorescencemicroscope. Cell express AP are GFP positive and cells do not express AP are GFPnegative ones.

Notes• Do not use pluripotent stem cell culture medium to dilute the 500x AP LiveSolution. Do not observe the result in the pluripotent stem cell culture medium.• Since AP is ubiquitously express in most cells types and highest in pluripotent stemcells. It is normal that dim staining can be observed in somatic cells such as CHOcells, HeLa cells and cells from placenta, and so on.• After removal of dye from the media, fluorescently labeled cells lose their signal byexocytosis in 1.5-2 hours, please observe the results within 1 hour after staining.• Please avoid repeated freezing and thawing. When necessary, the working concentration can be increased.Storage and HandlingStore at -20°C in a dark place. Avoid repeated freezing and thawing.FOR RESEARCH USE ONLYTel:

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