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Human Pluripotent Stem Cell Marker Antibody Panel Plus

Bio-Techne includes R&D Systems | Catalog # SC009

Key Product Details

Expression of Pluripotency Markers in Human Induced Pluripotent Stem Cells. 
(2)
Detection of SSEA-4 in Human Embryonic Stem Cells.

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

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FAQs for Human Pluripotent Stem Cell Marker Antibody Panel Plus

  • 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.

Product Documents for Human Pluripotent Stem Cell Marker Antibody Panel Plus

Certificate of Analysis

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