• Alkaline Phosphatase Live Solution (500X): 20 µl or 50 µl

• 24-well plate (or similar)
• DMEM/F12 preheated to 37 °C
• Pipettes and pipette tips
• Microcentrifuge tubes
• 37 °C CO₂ incubator
• Fluorescence microscope

The volumes of reagents in this protocol are based on a 24-well plate. For plates of other sizes, adjust the reagent volumes accordingly.

1. Remove the growth medium from the cultures.
2. Preheat DMEM/F12 to 37 °C. Wash with 500 µl of 37 °C DMEM/F12 for 3 minutes. Repeat the wash twice for a total of three times.
3. Prepare the 1X working solution by diluting the 500X Alkaline Phosphatase Live Solution with DMEM/F12.
4. Add 200 µl of 1X working solution directly to the cells and incubate in a 37 °C CO₂ incubator for 30 minutes.
5. Remove the working solution. Wash with 500 µl of DMEM/F12 for 3 minutes. Repeat the wash twice for a total of three times.
6. Add 500 µl of DMEM/F12 to the cells and observe the results under a fluorescence microscope. Cells that express alkaline phosphatase (AP) are GFP positive and cells that do not express AP are GFP negative.

1. Do not use pluripotent stem culture medium to dilute the 500X Alkaline Phosphatase Live Solution. Do not observe the result in pluripotent stem cell culture medium.
2. Alkaline phosphatase is ubiquitously expressed in most cell types and is highly expressed in pluripotent stem cells. It is normal to observe dim staining in some somatic cells such as CHO cells, HeLa cells, placental cells etc.
3. After removal of the dye from the media, fluorescently labelled cells lose their signal by exocytosis in 1.5-2 hours. It is recommended to observe the results within 1 hour after staining.
4. Avoid freeze/thaw cycles. The working concentration can be increased if necessary.