Creative Biolabs

Alkaline Phosphatase Staining Kit

[CAT#: NCC200551ZP]

A specific and sensitive tool for phenotypic assessment of ES/iPS cell differentiation by determining AP activity.

Stem Cell Culture
Cell Types:
Embryonic Stem Cell; iPSC

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Product Overview


The undifferentiated state of embryonic stem cells (ES) and induced pluripotent stem cells (iPS) can be characterized by high-level expression of alkaline phosphatase (AP), which together with the expression of other surface markers indicates that undifferentiated cells have The potential for self-differentiation-renewal. AP is a hydrolase enzyme that is responsible for dephosphorylating molecules such as nucleotides, proteins, and alkaloids under alkaline conditions. When an alkaline phosphatase staining kit is used to stain fixed ES or iPS cells, undifferentiated cells appear blue and differentiated cells appear colorless.

Cell Types

Embryonic Stem Cell; iPSC

Species Reactivity

Human; Mouse; Rat


Stem Cell Culture

Application Notes

Other materials required:
Fixative (eg 4% paraformaldehyde in PBS)

Research Areas

Stem Cell Research


AP Staining Solution A 10 ml
AP Staining Solution B 10 ml


Dry Ice



Handling Advices

A. Preparation of reagents

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

Shelf Life

This product is stable for 12 months when stored as directed.

Research Use Only

For research use only, not for diagnostic or therapeutic use.

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For Research Use Only. Not For Clinical Use.
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