Alkaline Phosphatase Staining

Pluripotent stem cell identification is a crucial process for establishing new induced pluripotent stem (iPS) cell lines and monitoring the quality of conventionally cultured embryonic stem (ES) cells and iPS cells, since the purity of iPS cells and their ability to differentiate directly affects the productivity and efficiency of cell expansion as well as the generation of terminally-differentiated cell types.

Alkaline phosphatase (AP) staining is a rapid method for screening pluripotent cells by using AP activity. AP is a hydrolase responsible for the dephosphorylation of many molecules, such as nucleotides, proteins, and alkaloids under alkaline conditions. AP activity is higher in pluripotent cells but is greatly decreased in more differentiated cell types. The technique described herein may be used to enumerate pluripotent cells during differentiation in the presence or absence of specific genetic manipulations or small chemical modulators. It may also be used to monitor induced pluripotency using defined factors from more differentiated cell types.

Materials and Equipment

  • Mouse embryonic stem cells (mESCs) (Creative-Bioarray, CSC-7878W)
  • 0.1% gelatin
  • Murine embryonic fibroblast (MEF) feeder cells
  • Alkaline Phosphatase Staining Kit (containing Fixation Solution, AP Staining Solution A, AP Staining Solution B) (Creative-Bioarray, CSK-ID001)
  • 1X PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2PO4, 0.24 g KH2PO4, add MilliQ H2O to a final volume of 1 L, pH 7.4)
  • 1X PBS-T (8 g NaCl, 0.2 g KCl, 1.44 g Na2PO4, 0.24 g KH2PO4, 500 µL Tween-20, add MilliQ H2O to a final volume of 1 L, pH 7.4)
  • 1X PBS-glycerol (8 g NaCl, 0.2 g KCl, 1.44 g Na2PO4, 0.24 g KH2PO4, 200 mL Glycerol, add MilliQ H2O to a final volume of 1 L, pH 7.4)

Assay Procedure

Cell culture

  • Coat plates with 0.1% gelatin. For each well of a 6 well plate, add 1.5 mL of 0.1% gelatin. Incubate the plates for 30 min at room temperature. Aspirate off the gelatin and allow to dry for 10 min prior to adding media and plating cells.
  • Seed each well with 4.75 x 105 murine embryonic feeder cells (MEFs) in 2 mL of media. Allow the MEFs to attach overnight prior to plating out the mouse ESCs.
  • Passage actively dividing cells and culture for 3-5 days to a subconfluent density prior to staining. For mouse ESCs (mESCs), approximately 1 x 105 cells are plated to each well of a 6-well plate (or 1 x 104 cells for 24-well plate) that has been previously coated with 0.1% gelatin and plated with murine embryonic fibroblast (MEF) feeder cells.

Alkaline phosphatase staining

  • Aspirate the culture media from each well and rinse with 1X PBS-T. 
  • Aspirate the rinse solution and fix the cells with Fixation Solution at room temperature for 2 min. Use a sufficient volume to endure complete coverage of the cells. This would be 500 µL for each well of 24-well plate or 2 mL for each well of a 6-well plate.

    Note: It is important not to exceed the fixation time of 2 min. Over fixation will result in the loss of AP activity and inaccurate results.

  • Aspirate the Fixation Solution and rinse with 1X PBS-T. The wells should not be allowed to dry out.
  • Prepare the Staining Solution by mixing Solution A and Solution B at a 1:1 ratio. To ensure that there is sufficient volume for staining.

    Note: The staining solution should be freshly prepared and used within 5 min of preparation.

  • Aspirate the 1X PBS-T and pipet on the prepared Staining Solution. Cover the plate with aluminum foil in the dark for approximately 15 min. Monitor the color change (pluripotent cells will stain red/purple, MEFs will remain colorless) to avoid non-specific staining.
  • Remove the staining solution by aspiration and wash the cells two times with 1X PBS.

    Note: Do not use 1X PBS-T, as this may wash out the staining.

  • Aspirate the 1X PBS. Add 1X PBS-glycerol for long-term storage at 4°C.

Enumeration of pluripotent cells

  • Count the number of AP stained (red/purple) pluripotent colonies and the differentiated colonies by eye or using a light microscope.


  1. Marti M. et al. Characterization of pluripotent stem cells. Nat Protoc, 2013, 8(2): 223-253. 
  2. Takahashi K. et al. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.

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