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Human Pluripotent Stem Cell Starter Kit

Catalog #: SC029 Datasheet / COA / SDS

Discontinued Product

SC029 has been discontinued.
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Human Pluripotent Stem Cell Starter Kit Summary

Kit Summary

Reagents to expand and characterize human pluripotent stem cells.

  • Offers a cost-effective way to expand and characterize pluripotent stem cells
  • Verifies the pluripotency of starting stem cell populations
  • Enables expansion of cells under feeder-free conditions
  • Provides an ideal setup kit for researchers who are new to the stem cell field

Why Use a Starter Kit for the Expansion and Characterization of Pluripotent Stem Cells?

Pluripotent stem cells can be efficiently expanded and characterized using specialized media, growth factors, and pluripotency marker antibodies. Although the techniques for expansion and characterization are well established, obtaining all of the necessary reagents can be expensive and time-consuming, especially for those just starting out in the field.   See Details

R&D Systems offers the Human Pluripotent Stem Cell Starter Kit, a cost-effective kit which contains reagents for the expansion and characterization of human pluripotent stem cells. The kit includes Mouse Embryonic Fibroblast (MEF) Conditioned Media, Human FGF basic, and Stem Cell Qualified Reduced Growth Factor (RGF) Basement Membrane Extract (BME), PathClear® to promote the expansion of undifferentiated cells under feeder-free conditions. To verify the pluripotency of the cells, the kit includes antibodies to Oct-4A, Nanog, and SSEA-4.

Kit Components

  • Mouse Embryonic Fibroblast (MEF) Conditioned Media
  • Human FGF basic (5000X) – enough to make 50 mL of a 5000X stock
  • Stem Cell Qualified Reduced Growth Factor (RGF) Basement Membrane Extract (BME), PathClear
  • Goat Anti-Human Nanog, 25 µg
  • Mouse Anti-Human Oct-4A,  25 µg
  • Mouse Anti-Human/Mouse SSEA-4, 25 µg

Stability and Storage

Store unopened kit at = -70 °C in a manual defrost freezer. This kit is stable for up to 3 months from the date of receipt.

 

Data Examples


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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 by immunofluorescence (A-C) and flow cytometry (D) using the indicated primary antibodies supplied in the Human Pluripotent Stem Cell Starter Kit (Catalog # SC029). The cells were stained using NorthernLights™ (NL)557-conjugated Donkey Anti-Goat Secondary Antibody (A; Catalog # NL001; red), NL557-conjugated Donkey Anti-Mouse Secondary Antibody (B; Catalog # NL007; red), NL493-conjugated Donkey Anti-Mouse Secondary Antibody (C; Catalog # NL009; green), PE-conjugated Goat Anti-Mouse Secondary Antibody (D; Catalog # F0102B; filled histogram) or Mouse IgG3 Isotype Control Antibody (D; Catalog # MAB007; open histogram). The nuclei were counterstained with DAPI in panels A-C. The data show that the cells express all three markers of pluripotency.

BG01V human embryonic stem cells are licensed from ViaCyte, Inc.

Specifications

Source
N/A
Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

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Product Datasheet
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Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, human pluripotent stem cells can be expanded and characterized using the following procedure

  • Coat plates with Stem Cell Qualified RGF BME PathClear®
  • Plate pluripotent cells in MEF Conditioned Media
  • Expand cells in culture
  • Characterize pluripotency marker expression
 
 

Reagents Provided

Reagents supplied in the Human Pluripotent Stem Cell Starter Kit (Catalog # SC029):

  • Mouse Embryonic Fibroblast (MEF) Conditioned Media
  • Human FGF basic (5000X) – enough to make 50 mL
  • Stem Cell Qualified Reduced Growth Factor (RGF) Basement Membrane Extract (BME), PathClear
  • Goat Anti-Human Nanog, 25 µg
  • Mouse Anti-Human Oct-4A,  25 µg
  • Mouse Anti-Human/Mouse SSEA-4, 25 µg

When antibodies are reconstituted at the recommended concentration of 1 μg/100 μL, there is enough of each to make 2.5 mL of staining solution.

Other Supplies Required

Reagents

  • Phosphate-Buffered Saline (PBS)
  • DMEM/F-12
  • Y-27632 dihydrochloride, p160ROCK inhibitor (Catalog # 1254)
  • Trypan Blue
  • Accutase™ (Innovative Cell Technologies, Catalog # AT-104 or equivalent)
  • BSA (very low endotoxin)
  • 4% Paraformaldehyde in PBS
  • 1% BSA in PBS
  • 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS (Blocking Buffer)
  • Flow Cytometry Staining Buffer (Catalog # FC001)
  • Mounting Medium (Catalog # CTS011)
  • Secondary antibody for immunocytochemistry (Catalog # NL001 and NL007)
  • Secondary antibody for flow cytometry (Catalog # F0102B)
  • Isotype control for flow cytometry (Catalog # MAB007)
  • Deionized or distilled water
  • 95% ethanol (EtOH)
 

Materials

  • Human pluripotent stem cells
  • 6-well culture plates
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 12 mm cover slips
  • 0.2 µm syringe filter
  • 10 mL syringes
  • Serological pipettes
  • Pipettes and pipette tips
  • Fine-pointed curved forceps
  • Glass slides
  • FACS™ tubes
 

Equipment

  • 37 °C, 5% CO2 incubator
  • 37 °C water bath
  • Clinical centrifuge
  • Hemocytometer
  • Inverted microscope
  • Fluorescence microscope
 

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Procedure Overview

    Coating Plates

  1. Add BME in DMEM/F-12 to plates.
  2. Incubate at room temperature for 1-2 hours.
  3. See Details
  4. Remove the media just prior to plating.

    5. Preparation & Plating of Human Pluripotent Stem Cells

  5. Transfer pluripotent stem cells to a 15 mL centrifuge tube containing pre-warmed MEF Conditioned Media.
  6. Centrifuge at 200 x g for 4 minutes.
  7. Resuspend the cell pellet in MEF Conditioned Media supplemented with FGF basic.
  8. Add the human pluripotent stem cells to the BME-coated plate
  9. Culture the cells at 37 °C and 5% CO2 and change the media daily.

    10. Cell Dissociation & Cell Plating

  10. Coat the plates with BME.
  11. Remove the media from cells.
  12. Add Accutase solution to each plate.
  13. Pipette gently over the plate until all of the cells have detached.
  14. Transfer the cell suspension to a 15 mL centrifuge tube containing = 5 mL of MEF Conditioned Media.
  15. Centrifuge at 200 x g for 4 minutes.
  16. Resuspend the pellet in MEF Conditioned Media.
  17. Perform a cell count.
  18. Plate the desired number of cells on BME-coated plates in MEF Conditioned Media containing FGF basic.
  19. Culture the cells at 37 °C and 5% CO2.
  20. Characterize cells by immunocytochemistry or flow cytometry.

    21. Immunocytochemistry

  21. Add a sterile coverslip to each well of a 24-well plate.
  22. Coat the wells with BME.
  23. See Details
  24. Plate pluripotent stem cells.
  25. Culture to desired density.
  26. Fix the stem cells with 4% paraformaldehyde.
  27. Wash the cells three times
  28. Permeabilize and block with blocking solution for 45 minutes at room temperature.
  29. Incubate with primary antibodies.
  30. Wash with wash buffer.
  31. Incubate with fluorochrome-conjugated secondary antibodies.
  32. Wash with wash buffer.
  33. Incubate with nuclear counterstain.
  34. Mount the coverslip.
  35. Visualize using a fluorescence microscope and appropriate filter sets.

    36. Flow Cytometry

  36. Perform a cell count on harvested cells.
  37. Resuspend the cells in FACS buffer at 1 x 106 cells/mL.
  38. See Details
  39. Transfer 90 µL of the cell suspension into a 5 mL flow cytometry tube.
  40. Add 10 µL of Anti-Human/Mouse SSEA-4 Antibody or an isotype control antibody.
  41. Incubate for 30 minutes at room temperature.
  42. Centrifuge samples at 300 x g for 5 minutes.
  43. Wash the cells three times with FACS buffer.
  44. Resuspend the cells in 200 µL FACS buffer.
  45. Add 10 µL of a fluorochrome-conjugated secondary developing reagent.
  46. Incubate for 30 minutes at room temperature in the dark.
  47. Centrifuge samples at 200 x g for 5 minutes.
  48. Wash the cells two times with FACS buffer.
  49. Resuspend the cells in 200 µL FACS buffer.
  50. Analyze the cells by flow cytometry.

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