Human Pluripotent Stem Cell Marker Antibody Panel
Human Pluripotent Stem Cell Marker Antibody Panel Summary
Five antibodies to verify human stem cell pluripotency:
Key Benefits
- A cost-effective selection of pluripotency marker antibodies
- Verifies the pluripotency of your starting stem cell population
- Increases confidence in pluripotency status by using 5 markers
Why Use Molecular Markers to Verify Human Stem Cell Pluripotency?
Stem cell pluripotency can be characterized functionally by their ability to differentiate into cells of the three vertebrate germ layers. However, functional verification is time-consuming, expensive, and is less conducive to routine stem cell culture screening or high-throughput analysis of stem cell pluripotency.
In addition to the abili ty to give rise to all cell types, a number of molecular markers have been identified to characterize the pluripotent status of stem cells. For example, human pluripotent stem cells express the cell surface proteins SSEA-4 and Alkaline Phosphatase and the transcription factors Oct-3/4 and Nanog.
Analyzing human stem pluripotency using marker antibodies:
- Promotes the identification and expansion of high quality, undifferentiated stem cell populations.
- Provides increased confidence in pluripotent status through the use of multiple markers.
- Defines the starting stem cell population to reduce experimental variation.
- Is a straightforward, cost-effective way to verify stem cell pluripotency.
This kit contains the following antibodies to verify human stem cell pluripotency:
Positive Markers
- Mouse Anti-Human Alkaline Phosphatase Monoclonal Antibody
- Goat Anti-Human Nanog Antigen-affinity Purified Polyclonal Antibody
- Goat Anti-Human Oct-3/4 Antigen-affinity Purified Polyclonal Antibody
- Mouse Anti-Human SSEA-4 Monoclonal Antibody
Negative Marker
- Mouse Anti-Human SSEA-1 Monoclonal Antibody
Each antibody is supplied as 25 µg; if reconstituted in 250 µL, this provides reagents sufficient for processing the equivalent of eight 300 µL immunocytochemistry samples or 25 flow cytometry samples at the recommended concentrations.
Stability and Storage
Reagents are stable for 12 months from date of receipt when stored in the dark at 2 °C to 8 °C.
Specifications
Product Datasheets
Scientific Data
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Expression of Pluripotency Markers in Human Embryonic Stem Cells. Pluripotency marker expression was detected in immersion-fixed BG01V human embryonic stem cells using antibodies supplied in the Human Pluripotent Stem Cell Marker Antibody Panel (Catalog # SC008). Pluripotency marker expression was analyzed by dual immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using Catalog # NorthernLights™ (NL)493- and NL557-conjugated Secondary Antibodies (green and red, respectively). Where indicated, the nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.
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Detection of Alkaline Phosphatase and SSEA-4 in Human Embryonic Stem Cells. Human embryonic stem cells were stained with pluripotency marker antibodies provided in the Human Pluripotent Stem Cell Marker Panel (Catalog # SC008).A.The cells were labeled with a Mouse Anti-Human Alkaline Phosphatase Monoclonal Antibody (filled histogram) or a Mouse IgG1Isotype Control Antibody (Catalog # MAB002; open histogram).B.The cells were also labeled with a Mouse Anti-Human SSEA-4 Monoclonal Antibody (filled histogram) or a Mouse IgG3Isotype Control Antibody (Catalog # MAB007; open histogram). The cells were stained using a PE-conjugated Goat Anti-Mouse Secondary Antibody (Catalog # F0102B).
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human stem cell pluripotency can be verified using marker antibodies and the following procedure:
- The cells are harvested and processed for either immunocytochemistry or flow cytometry
- The cells are incubated with antibody markers of pluripotency
- Pluripotency marker expression is analyzed
Reagents supplied in the Human Pluripotent Stem Cell Marker Antibody Panel Kit (Catalog # SC008):
Positive Markers
- Mouse Anti-Human Alkaline Phosphatase Monoclonal Antibody
- Goat Anti-Human Nanog Antigen-affinity Purified Polyclonal Antibody
- Goat Anti-Human Oct-3/4 Antigen-affinity Purified Polyclonal Antibody
- Mouse Anti-Human SSEA-4 Monoclonal Antibody
Negative Markers
- Mouse Anti-Human SSEA-1 Monoclonal Antibody
Immunocytochemistry
Reagents
- Appropriate stem cell culture substrate (e.g., StemXVivo® Culture Matrix (Catalog # NL001), iMEFs (Catalog # PSC001), etc.)
- Cell culture medium
- Sterile PBS
- 4% paraformaldehyde in PBS
- 1% BSA in PBS
- 0.1% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- Mounting medium (Catalog # CTS011 or equivalent)
- Secondary developing reagents (Catalog # NL001, NL003, NL007, NL009, or equivalent)
Materials
- Human pluripotent stem cells
- Cell culture plate (24-well)
Equipment
- 37 °C, 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Inverted microscope
- Fluorescence microscope
Flow Cytometry
Reagents
- Isotype controls (Catalog # MAB002 and MAB007, or equivalent)
- FACS buffer (2% fetal bovine serum, 0.1% sodium azide in Hank’s buffer)
- Secondary developing reagents
Materials
- Human pluripotent stem cells
- 5 mL tubes
Equipment
- 37 °C, 5% CO2 incubator
- 2 °C to 8 °C refrigerator
- Centrifuge
- Hemocytometer
- Flow cytometer
Triton is a registered trademark of Union Carbide, Inc.
Immunocytochemistry
Pass a suspension of mouse bone marrow cells through a 70 μm nylon strainer to remove clumps and debris.
Remove red blood cells if necessary.
Wash the cells with IMDM/2% FBS by centrifugation at 400 x g for 10 minutes and pool the cells.
Coat coverslips with stem cell subtype-specific substrate.
Plate stem cells.
Culture to desired density/age.
Fix stem cells with 4% paraformaldehyde.
Block with blocking solution.
Incubate with primary antibodies.
Wash with wash buffer.
Incubate with fluorochrome-conjugated secondary antibodies.
Wash with wash buffer.
Incubate with nuclear counterstain.
Mount the coverslip.
Visualize using a fluorescence microscope and appropriate filter sets.
Flow Cytometry
Perform a cell count on harvested cells.
Resuspend the cells in FACS buffer at 1 x 106 cells/mL.
Aliquot 90 µL of the cell suspension into a 5 mL flow cytometry tube.
Add 10 µL of each antibody or isotype control to the cells.
Incubate for 30 minutes at room temperature.
Centrifuge samples at 300 x g for 5 minutes.
Wash the cells three times with FACS buffer.
Resuspend the cells in 200 µL FACS buffer.
Add 10 µL of a fluorochrome-conjugated secondary developing reagent.
Incubate for 30 minutes at room temperature in the dark.
Centrifuge samples at 300 x g for 5 minutes.
Wash the cells three times with FACS buffer.
Resuspend the cells in 200 µL FACS buffer.
Analyze the cells by flow cytometry.
Citation for Human Pluripotent Stem Cell Marker Antibody Panel
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Hsa-miR-520d induces hepatoma cells to form normal liver tissues via a stemness-mediated process.
Authors: Tsuno S, Wang X, Shomori K, Hasegawa J, Miura N
Sci Rep, 2014-01-24;4(0):3852. 2014-01-24
FAQs
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What is the difference between the Human Embryonic Stem Cell Marker Antibody Panel (Catalog # SC008) and the Human Embryonic Stem Cell Marker Antibody Panel Plus (Catalog #SC009)?
Catalog # SC008 was developed with a panel of antibodies that were significant stem cell markers at the time of release. As research on embryonic stem cells progressed, the number of markers of interest increased. Catalog # SC009 was later introduced to include antibodies for newly discovered markers, in addition to the antibodies from the original panel.
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