Alkaline Phosphatase Staining Kit
A specific and sensitive tool for phenotypic assessment of ES/iPS cell differentiation by determining AP activity.
Fixative (eg 4% paraformaldehyde in PBS)
AP Staining Solution B 10 ml
Prepare enough AP staining solution for each experiment.
For one well of a 6-well plate, mix 0.5 ml AP staining solution A and 0.5 ml AP staining solution B in a 15 ml conical tube. For best results, the AP substrate solution should be used within 10 minutes of preparation.
Note: As long as the ratio of 1:1 is retained, you can enlarge or reduce the quantity in proportion. For example, for each well of a 24-well plate, 0.3 ml of AP substrate solution is recommended, and for a 12-well plate, 0.5 ml is recommended.
B. Staining of cells with alkaline phosphatase
The mouse/human stem cells were cultured for 5 days with or without feeder layers.
Aspirate the medium and wash the cells twice with 1 ml of 1X PBS. Aspirate the cleaning solution.
Add fixing solution to the cells, 0.5 ml per well for 24-well plates. Incubate at room temperature for 2 minutes.
Note: Do not over-fix the cells, as over-fixation may result in loss of AP activity.
Aspirate the fixative and wash the fixed cells twice with 1X PBS.
Remove 1X PBS and then add freshly prepared AP substrate solution to each well. For 24-well plates, add 0.3 ml per well.
Incubate the cells in the dark at room temperature for 10 to 20 minutes.
The reaction was stopped by pipetting the staining solution and rinsing the well twice with IX PBS.
Cover the cells with 1X PBS to prevent drying out.
Store the plate at 4°C