Human Pluripotent Stem Cell Marker Antibody Panel Plus
R&D Systems, part of Bio-Techne | Catalog # SC009
Key Product Details
Summary for Human Pluripotent Stem Cell Marker Antibody Panel Plus
Eight antibodies to verify human stem cell pluripotency.
Key Benefits
- 8 markers for increased confidence in pluripotency status
- Verifies the pluripotency of your starting stem cell population
- Cost-effective versus purchasing individual pluripotency marker antibodies
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 ability 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 CD9 Monoclonal Antibody
- Mouse Anti-Human E-Cadherin 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 PODXL (GCTM antigen) Monoclonal Antibody
- Mouse Anti-Human SSEA-4 Monoclonal Antibody
- Mouse Anti-Human SOX2 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 eight 300 µL immunocytochemistry samples or 25 flow cytometry samples.
Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.
R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.
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Expression of Pluripotency Markers in Human Induced Pluripotent Stem Cells. iPS2 human induced pluripotent stem cells were cultured on Irradiated Mouse Embryonic Fibroblasts (iMEFs; Catalog PSC001) and labeled with antibodies from the Human Pluripotent Stem Cell Marker Antibody Panel Plus (Catalog # SC009). Pluripotency marker expression was analyzed by dual immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using NorthernLights™ (NL)486- 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 SSEA-4 in Human Embryonic Stem Cells. Human embryonic stem cells were stained with the SSEA-4 Monoclonal Antibody provided in the Human Pluripotent Stem Cell Marker Panel Plus (Catalog # SC009; 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
- 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 Plus Kit (Catalog # SC009):
Positive Markers
- Mouse Anti-Human CD9 Monoclonal Antibody
- Mouse Anti-Human E-Cadherin 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 PODXL (GCTM antigen) Monoclonal Antibody
- Mouse Anti-Human SSEA-4 Monoclonal Antibody
- Mouse Anti-Human SOX2 Monoclonal Antibody
Negative Marker
- Mouse Anti-Human SSEA-1 Monoclonal Antibody
Immunocytochemistry
Reagents
- Appropriate stem cell culture substrate (e.g., StemXVivo® Culture Matrix (Catalog # CCM013), iMEFs (Catalog # PSC001), etc.)
- Cell culture medium
- Sterile PBS
- 4% paraformaldehyde in PBS
- 1% BSA in PBS
- 0.3% 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)
- Flow cytometry staining buffer (Catalog # FC001)
- Secondary developing reagents (Catalog # F0101B, F0102B, F0103B, F0114, F0116, F0117, F0118, F0119, or equivalent)
- Sterile PBS
Materials
- Human pluripotent stem cells
- 5 mL tubes
Equipment
- 37 °C, 5% CO2 incubator
- Centrifuge
- Hemocytometer
- Flow cytometer
Immunocytochemistry
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.
Flow Cytometry
Perform a cell count on harvested.
Resuspend cells in Flow Cytometry Staining Buffer at a concentration of 1 x 106 cells/1 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.
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