Alkaline Phosphatase Staining Kit
[CAT#: NCC200551ZP]
A specific and sensitive tool for phenotypic assessment of ES/iPS cell differentiation by determining AP activity.
- Applications:
- Stem Cell Culture
- Cell Types:
- Embryonic Stem Cell; iPSC
Certificate of Analysis Lookup
To download a Certificate of Analysis, please enter a lot number in the search box below. Note: Certificate of Analysis not available for kit components.
Lot Number
To download a Certificate of Analysis, please enter a lot number in the search box below. Note: Certificate of Analysis not available for kit components.
Lot Number
Product Overview
Description
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
Applications
Stem Cell Culture
Application Notes
Other materials required:
Fixative (eg 4% paraformaldehyde in PBS)
1X PBS
Fixative (eg 4% paraformaldehyde in PBS)
1X PBS
Research Areas
Stem Cell Research
Properties
Components
AP Staining Solution A 10 ml
AP Staining Solution B 10 ml
AP Staining Solution B 10 ml
Shipping
Dry Ice
Storage
4°C
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
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.
Neural Cell Culture
For Research Use Only. Not For Clinical Use.
