StemTAG™ Alkaline Phosphatase Activity Assay Kit, Colorimetric

Catalog No. ABIN2344931

Product Details

Reactivity: Human

Detection Method: Fluorometric, Colorimetric

Application: Activity Assay (AcA)

Brand: StemTAG™

Sample Type: Cell Samples

Components:

1. StemTAG™ AP Activity Assay Substrate : One bottle - 5 mL
2. Cell Lysis Buffer : One bottle - 20 mL
3. 10X Stop Solution : One bottle - 10 mL
4. AP Activity Assay Standard : One tube - 1 mL of 5 mM p-Nitrophenol

Material not included:

1. Human or Mouse Embryonic Stem Cells and Culture Medium
2. 1X PBS
3. 1X PBST (1X PBS containing 0.05 % Tween-20)
4. Deionized Water
5. Light Microscope
6. 96-well Microplate Reader

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Measure the ubiquitous alkaline phosphatase marker in embryonic stem cells and embryonic germ cells
• Quantify activity by colorimetric or fluorometric plate reader

Reagent Preparation:

• 1X Stop Solution: Prepare a 1X Stop Solution by diluting the provided 10X stock 1:10 in deionized water. Store the diluted solution at room temperature.

Assay Procedure:

1. Culture mouse ES cells in medium containing LIF, alternatively, culture human ES cells on a MEF feeder layer.
2. Gently aspirate the medium from the ES cells and wash the cells twice with cold PBS. Aspirate the wash solutions.
3. Lyse the cells in Cell Lysis Buffer (0.5 mL for a 35 mm dish).
4. Incubate for 10 minutes at 4 °C, remove the solution and spin down the cell debris at 12,000 X g for 10 minutes. Save the supernatant as cell lysate. Perform a BCA assay or other protein assay to determine the protein concentration of the cell lysate.
5. Add 50 μL of cell lysate to a 96-well plate. In addition, prepare blank wells that contain 50 μL Cell Lysis Buffer. We recommend testing samples in triplicate.
6. Initiate the reaction by adding 50 μL of StemTAG™ AP Activity Assay Substrate. Incubate for 10-30 minutes at 37 °C.
7. Stop the reaction by adding 50 μL of 1X Stop Solution and mix by placing the plate on an orbital plate shaker for 30 seconds.
8. Read the absorbance of each well at 405 nm.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store all components at 4°C.

References

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Target Details

Background: Embryonic stem (ES) cells are continuous proliferating stem cell lines of embryonic origin first isolated from the inner cell mass (ICM). Two distinguishing features of ES cells are their ability to be maintained indefinitely in an undifferentiated state and their potential to develop into any cell within the body. Based on previous methods developed for mouse ES cells, human ES cell lines were first established by Dr. James Thomson and colleagues. Like mouse ES cells, human ES cells express high levels of membrane alkaline phosphatase (AP) and Oct-4, a transcriptional factor critical to ICM and germline formation. However, unlike mouse ES cells, hES cells do not express stage-specific embryonic antigen (SSEA-1). In addition, prolonged propagation of hES cells is typically achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Human ES cell lines are not able to maintain their undifferentiated state in the absence of supporting feeder layer cells, even when exogenous cytokines such as leukemia inhibitory factor (LIF) and gelatin-coated plates are used.