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Rat Cortical Stem Cells (3 x 10e6 cells/vial)

Rat Cortical Stem Cells (3 x 10^6 cells/vial) (Catalog # NSC001)
(4)
Verification of Neural Progenitor Cell Multipotency Following Expansion with N-2 MAX Media Supplement.
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Neural Progenitor Cells Expanded with N-2 Plus Media Supplement Express Nestin and SOX2.
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Differentiation of Rat Cortical Stem Cells.
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Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Rat Cortical Stem Cells can be thawed and expanded by the neurosphere or monolayer system:

  • Thaw Rat Cortical Stem Cells
  • Plate cells in Completed NSC Base Media containing FGF basic
  • Expand cells using the monolayer or neurosphere system
 

Reagents & Materials

Reagents Supplied in Rat Cortical Stem Cells (Catalog # NSC001)

  • 1 vial of Rat Cortical Stem Cells containing 3 x 106 cells.

Other Supplies Required

Expanding Rat Cortical Stem Cells Using the Neurosphere System

Reagents

  • N-2 Plus Media Supplement (Catalog # AR003)
  • Recombinant FGF basic (Catalog # 233-FB)
  • Bovine Fibronectin (Catalog # 1030-FN)
  • PBS
  • DMEM/F12
  • Glucose
  • Glutamine
  • NaHCO3
  • Penicillin-Streptomycin 100X
  • Poly-L-ornithine
  • Ca2+/Mg2+-free Hank's Balanced Salt Solution (HBSS) (10X)
  • HEPES
  • BSA, very low endotoxin
  • Trypan Blue, 0.4%
  • Deionized (DI) water
 

Materials

  • 10 cm tissue culture plates
  • 50 mL centrifuge tubes
  • 0.2 µm, sterile filter units
  • Plastic cell scraper
  • Pipettes and pipette tips

Equipment

  • 37 °C and 5% incubator
  • Centrifuge
  • Hemocytometer
  • Microscope

Other Supplies Required

Expanding Rat Cortical Stem Cells Using the Monolayer System

Reagents

  • N-2 Plus Media Supplement (Catalog # AR003) or N-2 MAX Media Supplement (Catalog # AR009)
  • Recombinant FGF basic (Catalog # 233-FB)
  • Bovine Fibronectin (Catalog # 1030-FN)
  • PBS
  • DMEM/F12
  • Glucose
  • Glutamine
  • NaHCO3
  • BSA, very low endotoxin
  • Trypan Blue, 0.4%
  • Acetic Acid
  • Deionized (DI) water
 

Materials

  • 6-well plates
  • 15 mL centrifuge tubes
  • Pasteur pipettes
  • Pipettes and pipette tips
  • 0.2 μm, sterile filter unit

Equipment

  • 37 °C and 5% incubator
  • Centrifuge
  • Hemocytometer
  • Microscope

Procedure Overview for Expanding Rat Cortical Stem Cells Using the
Neurosphere System

Thawing Cryopreserved Rat Cortical Stem Cells

    Add 1 mL of fresh pre-warmed media to the vial of frozen rat cortical stem cells
  1. Add 1 mL of fresh pre-warmed media to the vial of frozen rat cortical stem cells.
  2. See Details
  3. Pipette up and down and as cells thaw, transfer the thawed portion into a 50 mL tube containing pre-warmed Completed Base NSC Media supplemented with 20 ng/mL FGF basic.
  4. Centrifuge the cells at 200 x g for 5 minutes.
  5.  

    Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing FGF basic
  6. Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing FGF basic.
  7.  

    Neurosphere Expansion

    Perform a cell count
  8. Perform a cell count.
  9.  

    Plate cells at approximately 1.0 x 10<sup>6</sup> NSCs in 5 mL of Completed NSC Base Media supplemented with FGF basic per well in a 6-well plate
  10. Plate cells at approximately 1.0 x 106 NSCs in 5 mL of Completed NSC Base Media supplemented with FGF basic per well in a 6-well plate.
  11. Incubate the cells at 37 °C and 5% CO2.
  12. Add fresh FGF basic to the media daily.
  13. &nbsp;

    Replace the media every 4 days according to the number of neurospheres present
  14. Replace the media every 4 days according to the number of neurospheres present:
    1. a. Less than 50 neurospheres:
      1. &bull; Transfer the neurospheres into one well of a 6-well plate using 2.5 mL of fresh media and 2.5 mL of spent/conditioned media.
    2. b. More than 50 neurospheres:
      1. &bull; Transfer the media containing the neurospheres to a 15 mL tube.
      2. &bull; Centrifuge for 5 minutes at 100 x g and remove the media.
      3. &bull; Resuspend the pellet using a small quantity of fresh Completed NSC Base Media containing FGF basic.
      4. &bull; Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing FGF basic in one well of a 6-well plate.
  15. Passage the cells at 5-6 days or when the neurospheres have a dark clump inside or ruffling on the outside.
  16. &nbsp;

    Passing Neurosphere

    Transfer the media containing the floating neurospheres to a 15 mL tube
  17. Transfer the media containing the floating neurospheres to a 15 mL tube.
  18. Centrifuge for 5 minutes at 100 x g.
  19. &nbsp;

    Partially dissociate the neurospheres by pipetting up and down 20 times with a P200 pipette
  20. Partially dissociate the neurospheres by pipetting up and down 20 times with a P200 pipette.
  21. At passage 1 and 2 the cells should be split 1:1. After passage 2 the cells can be split 1:2.
  22. &nbsp;

Procedure Overview for Expanding Rat Cortical Stem Cells Using the
Monolayer System

Thawing Cryopreserved Mouse Cortical Stem Cells

    Coat cell culture plates with Poly-L-ornithine and Fibronectin.
  1. Coat cell culture plates with Poly-L-ornithine and Fibronectin.
  2. &nbsp;
  3. See&nbsp;Details
  4. Add 1 mL of fresh pre-warmed media to the vial of frozen rat cortical stem cells.

  5. Pipette up and down and as cells thaw.
  6. Pipette up and down and as cells thaw.
  7. Transfer the thawed portion into a 50 mL tube containing pre-warmed Completed Base NSC Media supplemented with 20 ng/mL FGF basic.
  8. &nbsp;

    Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan Blue
  9. Mix 10 &mu;L of the cell suspension with 10 &mu;L of 0.4% Trypan Blue
  10. Perform a cell count.
  11. &nbsp;

    Monolayer Expansion

    Plate 1.0 -1.5 x 10<sup>6</sup> NSCs in 10 mL of Completed NSC Base Media supplemented with FGF basic onto a Poly-L-ornithine/Fibronectin-coated 10 cm plate
  12. Plate 1.0 -1.5 x 106 NSCs in 10 mL of Completed NSC Base Media supplemented with FGF basic onto a Poly-L-ornithine/Fibronectin-coated 10 cm plate.
  13. Incubate the cells at 37 °C and 5% CO2.
  14. &nbsp;

    Replace the media once cells become adherent. After 24 hours, add 10 µL of FGF basic stock (1000X) to the culture
  15. Replace the media once cells become adherent. After 24 hours, add 10 &mu;L of FGF basic stock (1000X) to the culture.
  16. Replace the media with fresh Completed NSC Base Media every second day.
  17. Supplement the media daily with FGF basic.
  18. Passage the cells when they reach 60-70% confluency.
  19. &nbsp;

    Passaging Cells

    Wash the cells once with pre-warmed HBSS
  20. Wash the cells once with pre-warmed HBSS.
  21. Add 5 mL of HBSS.
  22. Incubate at room temperature until the cells round up.
  23. &nbsp;

    Scrape the cells from the plate
  24. Scrape the cells from the plate.
  25. Transfer the cells to a 50 mL centrifuge tube.
  26. Centrifuge for 5 minutes at 200 x g.
  27. Remove the supernatant.
  28. &nbsp;

    Resuspend the cells in 5 mL of Completed NSC Base Media containing FGF basic
  29. Resuspend the cells in 5 mL of Completed NSC Base Media containing FGF basic.
  30. &nbsp;

    Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan Blue
  31. Mix 10 &mu;L of the cell suspension with 10 &mu;L of 0.4% Trypan Blue
  32. Perform a cell count.
  33. &nbsp;

    Plate 0.8-1.0 x 106 viable cells in 10 mL of Completed NSC Base Media containing FGF basic onto a Poly-L-ornithine/Fibronectin-coated plate
  34. Plate 0.8-1.0 x 106 viable cells in 10 mL of Completed NSC Base Media containing FGF basic onto a Poly-L-ornithine/Fibronectin-coated plate.
  35. Incubate the cells at 37 °C and 5% CO2.
  36. &nbsp;

    Replace the media with fresh Completed NSC Base Media every second day
  37. Replace the media with fresh Completed NSC Base Media every second day.
  38. Supplement the media with FGF basic daily.
  39. Passage the cells after 3 days or when the cells reach 70% confluency.
  40. &nbsp;
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