StemTAG™ Alkaline Phosphatase Staining Kit (Red)

Catalog No. ABIN2344930

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 A : One amber bottle - 20 mL
3. StemTAG™ AP Staining Solution B : One amber bottle - 20 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

Reagent Preparation:

• StemTAG™ AP Staining Solution: Prepare FRESH 1X StemTAG™ AP Staining Solution by mixing equal volume of StemTAG™ AP Staining Solution A and StemTAG™ AP Staining Solution B. The volume of StemTAG™ AP Staining Solution needed is based on the number of samples. The chart below is suggested for samples in a 24-well plate, and may be modified accordingly to suit other culture plate sizes. Reagents Half plate (12 samples) 1 plate (24 samples) 4 plates (96 samples) Staining Solution A 2.4 mL 4.8 mL 9.6 mL Staining Solution B 2.4 mL 4.8 mL 9.6 mL Total 4.8 mL 9.6 mL 19.2 mL Table 2. Preparation of StemTAG™ AP Staining Solution 3

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 freshly prepared StemTAG™ AP Staining Solution (see Preparation of Reagents section).
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 red stained cell colonies (undifferentiated ES cells) vs. colorless colonies (differentiated ES cells) using a light microscope.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store all components at 4°C.

References

Lee, Lee, Kim, Park, Do, Kim, Choi, Kim, Song: "Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes." in: Stem cells international, Vol. 2016, pp. 6029271, (2016) (PubMed).

Lee, Lee, Do, Park, Kim, Kim, Chung, Kim, Song: "In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells." in: Cellular reprogramming, Vol. 18, Issue 4, pp. 246-55, (2016) (PubMed).

Jacinto, Benner, Hetzer: "The nucleoporin Nup153 regulates embryonic stem cell pluripotency through gene silencing." in: Genes & development, Vol. 29, Issue 12, pp. 1224-38, (2015) (PubMed).

Langlois, da Costa Reis Monte-Mor, Lenglet, Droin, Marty, Le Couédic, Almire, Auger, Mercher, Delhommeau, Christensen, Helin, Debili, Fuks, Bernard, Solary, Vainchenker, Plo: "TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells." in: Stem cells (Dayton, Ohio), Vol. 32, Issue 8, pp. 2084-97, (2014) (PubMed).

Manukyan, Singh: "Epigenome rejuvenation: HP1? mobility as a measure of pluripotent and senescent chromatin ground states." in: Scientific reports, Vol. 4, pp. 4789, (2014) (PubMed).

Lee, Lee, Lee, Kim, Chung, Kim, Kim, Choi, Kim, Song: "Identification and in vitro derivation of spermatogonia in beagle testis." in: PLoS ONE, Vol. 9, Issue 10, pp. e109963, (2014) (PubMed).

Lee, Lee, Jung, Kim, Roh, Jung, Park: "Ultraviolet A regulates adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells via up-regulation of Kruppel-like factor 2." in: The Journal of biological chemistry, Vol. 285, Issue 42, pp. 32647-56, (2010) (PubMed).

Izadyar, Pau, Marh, Slepko, Wang, Gonzalez, Ramos, Howerton, Sayre, Silva: "Generation of multipotent cell lines from a distinct population of male germ line stem cells." in: Reproduction (Cambridge, England), Vol. 135, Issue 6, pp. 771-84, (2008) (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.