StemTAG™ Alkaline Phosphatase Staining Kit (Purple)

Catalog No. ABIN2344933

Product Details

Reactivity: Human

Application: Staining Methods (StM)

Brand: StemTAG™

Sample Type: Cell Samples

Components:

1. Fixing Solution : One bottle - 50 mL
2. StemTAG™ AP Staining Solution : One amber bottle - 40 mL

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

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
• Detect by ICC staining

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 with 1 mL of 1X PBST. Aspirate the wash solution.
3. Add Fixing Solution to the cells, 0.4 mL per well for a 24-well plate. Incubate at room temperature for 2 minutes.
4. Remove the fixing solution and wash the fixed cells twice with 1 mL of 1X PBST.
5. Aspirate the final wash, and add 0.4 mL per well of StemTAG™ AP Staining Solution
6. Incubate the cells at room temperature for 15-30 minutes, protected from light.
7. Remove the AP Staining Solution, and then wash the stained cells twice with 1 mL of 1X PBS. Store cells in 1X PBS at 4 °C. For long-term storage, overlay the cells with 1X PBS containing 20 % Glycerol. Store at 4 °C.
8. Count the purple stained cell colonies (undifferentiated ES cells) vs. colorless colonies (differentiated ES cells) using a light microscope. 3

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: Store all components at 4°C.

References

Khan, Czarnecka, Lewicki, Helbrecht, Brodaczewska, Koch, Zdanowski, Król, Szczylik: "Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells." in: PLoS ONE, Vol. 11, Issue 11, pp. e0165718, (2016) (PubMed).

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.