Human Pluripotent Stem Cell Marker Antibody Panel Plus
R&D Systems, part of Bio-Techne | Catalog # SC009
Key Product Details
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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