Order Information
Catalog# Size Price
AP01 50 assays $99
 
Overview
Product Description cat. no. Format
ALSTEM Alkaline Phosphatase Staining Kit AP01 1 kit
Components Size Storage
AP Staining Solution A 10 ml 4°C
AP Staining Solution B 10 ml 4°C
 
Description
The undifferentiated state of embryonic stem (ES) and induced pluripotent stem (iPS) cells can be characterized by a high level of alkaline phosphatase (AP) expression which, along with the expression of other surface markers, indicates undifferentiated cells with the potential to self-renew. AP is a hydrolase enzyme responsible for dephosphorylating molecules such as nucleotides, proteins, and alkaloids under alkaline conditions. When fixed ES or iPS cells are stained using the Alkaline Phosphatase Staining Kit, undifferentiated cells appear blue, whereas differentiated cells appear colorless. ALSTEM's Alkaline Phosphatase Staining Kit is a specific and sensitive tool for the phenotypic assessment of ES/iPS cell differentiation by the determination of AP activity.
 
Additional Materials Required
Fixative (e.g. 4% paraformaldehyde in PBS)
1X PBS
Procedure

A. Preparation of Reagents

  • Prepare sufficient AP staining solution for each experiment.
  • For one well of 6-well plate, mix 0.5 ml of AP Staining Solution A and 0.5 ml of AP Staining Solution B in a 15 ml conical tube. For optimal results, the AP substrate solution should be used within 10 minutes after preparation.
Note: Quantities can be scaled up or down, as long as a 1:1 ratio is preserved. For example, 0.3 ml of AP substrate solution is suggested for each well of 24-well plate and 0.5 ml for 12-well plate.

B. Alkaline Phosphatase Staining of Cells

  1. Culture mouse/human stem cells on feeder layer or feeder-free for 5 days.
  2. Aaspirate the culture medium and wash the cells with 1 ml of 1X PBS twice. Aspirate the wash solution.
  3. Add Fix Solution to the cells, 0.5 ml per well for a 24-well plate. Incubate at room temperature for 2 min.
  4. Note: Do not over fix the cells, as over-fixation may result in the loss of AP activity.
  5. Aspirate the Fix Solution and wash the fixed cells with 1X PBS twice.
  6. Remove the 1X PBS and add freshly prepared AP Substrate Solution into each well. For a 24-well plate, add 0.3 ml per well.
  7. Incubate the cells in the dark at room temperature for 10 to 20 minutes.
  8. Stop the reaction by aspirating the staining Solution and rinsing the wells twice with 1X PBS.
  9. Cover the cells with 1X PBS to prevent drying out.
  10. Store the plate at 4 °C