Human Pluripotent Stem Cell Marker Antibody Panel Plus

Catalog # Availability Size / Price Qty
SC009
Expression of Pluripotency Markers in Human Induced Pluripotent Stem Cells. 
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Product Details
Procedure
Citations (2)
FAQs
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Human Pluripotent Stem Cell Marker Antibody Panel Plus Summary

Kit Summary

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.
 

 

Kit Contents

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.

 

 

Data Examples
Expression of Pluripotency Markers in Human Induced Pluripotent Stem Cells
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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) (R&D Systems, Catalog # PSC001) and labeled with antibodies from this kit. Pluripotency marker expression was analyzed by dual immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using NorthernLights™ (NL)493- and NL557-conjugated Secondary Antibodies (green and red, respectively). Where indicated, the nuclei were counterstained with DAPI (blue).

Expression of Expression of Pluripotency Markers in Embryonic and Induced Pluripotent Stem Cells.
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Expression of Pluripotency Markers in Embryonic and Induced Pluripotent Stem Cells. A,B. iBJ6 human induced pluripotent stem cells were cultured in mTeSR™. C. D3 mouse embryonic stem cells were cultured in StemXVivo® Mouse Pluripotent Stem Cell Media (R&D Systems, Catalog # CCM025). D,E. BG01V human embryonic stem cells were cultured in MEF Conditioned Media (R&D Systems, Catalog # AR005). Cultured cells were then labeled with antibodies provided in this kit. Pluripotency marker expression was analyzed by dual (A,B) or single (C) immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using NorthernLights™ (NL)493- and NL557-conjugated Secondary Antibodies (green and red, respectively) and counterstained with DAPI (blue).

Detection of SSEA-4 in Human Embryonic Stem Cells
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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 IgG3 Isotype Control Antibody (Catalog # MAB007; open histogram). The cells were stained using a PE-conjugated Goat Anti-Mouse Secondary Antibody (Catalog # F0102B).

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.

Specifications

Shipping Conditions
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Species
Human

Product Datasheets

Data Example

Immunocytochemistry Expression of Pluripotency Markers in Human Induced Pluripotent Stem Cells.  View Larger

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.

Flow Cytometry Detection of SSEA-4 in Human Embryonic Stem Cells. View Larger

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 Provided

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

 

Other Supplies Required

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

Materials

  • Human pluripotent stem cells
  • 5 mL tubes

Equipment

  • 37 °C, 5% CO2 incubator
  • Centrifuge
  • Hemocytometer
  • Flow cytometer

 

Procedure Overview

Immunocytochemistry

Coat coverslips with stem cell subtype-specific substrate.

Coat coverslips with stem cell subtype-specific substrate

Plate stem cells.

Culture to desired density/age.

Plate stem cells

Fix stem cells with 4% paraformaldehyde.

Fix stem cells with 4% paraformaldehyde

Block with blocking solution

Block with blocking solution

Incubate with primary antibodies.

Wash with wash buffer.

Incubate with primary antibodies

Incubate with fluorochrome-conjugated secondary antibodies.

Wash with wash buffer.

Incubate with fluorochrome-conjugated secondary antibodies

Incubate with nuclear counterstain.

Incubate with nuclear counterstain

Mount the coverslip.

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.

Incubate with nuclear counterstain

Aliquot 90 µL of the cell suspension into a 5 mL flow cytometry tube.

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.

Add 10 µL of each antibody or isotype control to the cells

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.

Centrifuge samples at 300 x g for 5 minutes

Add 10 µL of a fluorochrome-conjugated secondary developing reagent.

Incubate for 30 minutes at room temperature in the dark.

Add 10 µL of a fluorochrome-conjugated secondary developing reagent

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.

Centrifuge samples at 300 x g for 5 minutes

Analyze the cells by flow cytometry.

Analyze the cells by flow cytometry

Citations for Human Pluripotent Stem Cell Marker Antibody Panel Plus

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.

2 Citations: Showing 1 - 2
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  1. The development of cutaneous neurofibromas.
    Authors: Jouhilahti EM, Peltonen S, Callens T, Jokinen E, Heape AM, Messiaen L, Peltonen J
    Am. J. Pathol., 2012;178(2):500-5.  2012
  2. The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used.
    J. Cell. Biochem., 2009;106(5):867-77.  2009

FAQs

  1. 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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