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Mouse Cortical Stem Cells (2 x 10e6 cells/vial)

Key Product Details

Mouse Cortical Stem Cells (2 x 10e6 cells/vial)
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NSC002

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Mouse Cortical Stem Cells can be thawed and expanded by the neurosphere or monolayer system using the following procedure:

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

Reagents Provided

Reagents Supplied in Mouse Cortical Stem Cells (Catalog # NSC002):

  • 2 vials of Mouse Cortical Stem Cells containing 2 x 106 cells

Other Supplies Required

Expanding Mouse Cortical Stem Cells Using the Neurosphere System

Reagents

  • Mouse Cortical Stem Cells (Catalog # NSC002)
  • N-2 Plus Media Supplement (Catalog # AR003)
  • Recombinant Human Fibroblast Growth Factor basic (FGF basic) (Catalog # 233-FB or 4114-TC)
  • Recombinant Human Epidermal Growth Factor (EGF) (Catalog # 236-EG)
  • PBS
  • DMEM/F12
  • Glucose
  • Glutamine
  • NaHCO3
  • Penicillin-Streptomycin, 100X
  • BSA, very low endotoxin
  • Acetic acid
  • Trypan Blue
  • Deionized water
 

Materials

  • 10 cm tissue culture dishes
  • 15 mL tubes
  • 50 mL Falcon tubes
  • 0.2 µm, 1000 mL filter unit
  • 0.2 µm, 500 mL filter unit
  • Plastic cell scraper
  • Pipettes and pipette tips
 

Equipment

  • 37 °C and 5% CO2 incubator
  • Centrifuge
  • Hemocytometer
  • Microscope
  • Water bath
 

Other Supplies Required

Expanding Mouse Cortical Stem Cells Using the Monolayer System

Reagents

  • Mouse Cortical Stem Cells (Catalog # NSC002)
  • N-2 Plus Media Supplement (Catalog # AR003)
  • Recombinant Human Fibroblast Growth Factor basic (FGF basic) (Catalog # 233-FB or 4114-TC)
  • Recombinant Human Epidermal Growth Factor (EGF) (Catalog # 236-EG)
  • Purified Bovine Fibronectin (Catalog # 1030-FN)
  • PBS
  • DMEM/F12
  • Glucose
  • Glutamine
  • NaHCO3
  • Poly-L-ornithine
  • Hank’s Balanced Salt Solution (HBSS) (Ca2+/Mg2+-free), 10X
  • HEPES
  • BSA, very low endotoxin
  • Acetic acid
  • Trypan Blue
  • Deionized (DI) water
 

Materials

  • 10 cm tissue culture dishes
  • 15 mL tubes
  • 50 mL Falcon tubes
  • Pipettes and pipette tips
  • 0.2 µm, 1000 mL filter unit
  • 0.2 µm, 500 mL filter unit
  • Plastic cell scraper
 

Equipment

  • 37 °C and 5% CO2 incubator
  • Centrifuge
  • Hemocytometer
  • Microscope
  • Water bath
 

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

Thawing Cryopreserved Mouse Cortical Stem Cells

    Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells
  1. Add 1 mL of fresh pre-warmed media to the vial of frozen mouse 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 EGF and 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 EGF and FGF basic
  6. Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing EGF and FGF basic.
  7.  

    Neurosphere Expansion

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

    Plate cells at approximately 2.0 x 105 NSCs in 5 mL of Completed NSC Base Media supplemented with EGF and FGF basic per well in a 6-well plate
  10. Plate cells at approximately 2.0 x 105 NSCs in 5 mL of Completed NSC Base Media supplemented with EGF and FGF basic per well in a 6-well plate.
  11. Incubate the cells at 37 °C and 5% CO2.
  12. Add fresh EGF and FGF basic daily to the media.
  13.  

    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. • 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. • Transfer the media containing the neurospheres to a 15 mL tube.
      2. • Centrifuge for 5 minutes at 100 x g and remove the media.
      3. • Resuspend the pellet using a small quantity of fresh Completed NSC Base Media containing EGF and FGF basic.
      4. • Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing EGF and FGF basic in one well of a 6-well plate.
  15. Passage the cells at 5-7 days or when the neurospheres have a dark clump inside or ruffling on the outside.
  16.  

    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.  

    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 passages 1 and 2 the cells should be split 1:1. After passage 2 the cells can be split 1:2.
  22.  

Procedural Overview for Expanding Mouse 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. See Details
  3. Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells.
  4. 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 EFG and 20 ng/mL FGF basic.
  5.  

    Mix 10 µL of the cell suspension with 10 µL of 0.4% Trypan Blue
  6. Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
  7. Perform a cell count.
  8.  

    Monolayer Expansion

    Plate 2.0 x 10<sup>6</sup> NSCs in 10 mL of Completed NSC Base Media supplemented with EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated 10 cm plate
  9. Plate 2.0 x 106 NSCs in 10 mL of Completed NSC Base Media supplemented with EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated 10 cm plate.
  10. Incubate the cells at 37 °C and 5% CO2.
  11. &nbsp;

    Replace the media once cells become adherent. After 24 hours, add 10 µL of FGF basic stock (1000X) and 10 µL of EGF stock (1000X) to the culture
  12. Replace the media once cells become adherent. After 24 hours, add 10 &mu;L of FGF basic stock (1000X) and 10 &mu;L of EGF stock (1000X) to the culture.
  13. Replace the media with fresh Completed NSC Base Media every second day.
  14. Supplement the media daily with EGF and FGF basic
  15. Passage the cells when they reach 70-80% confluency.
  16. &nbsp;

    Passaging Cells

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

    Scrape the cells from the plate
  21. Scrape the cells from the plate.
  22. Transfer the cells to a 15 mL centrifuge tube.
  23. Centrifuge for 5 minutes at 200 x g.
  24. Remove the supernatant.
  25. &nbsp;

    Resuspend the cells in 5 mL of Completed NSC Base Media containing EGF and FGF basic
  26. Resuspend the cells in 5 mL of Completed NSC Base Media containing EGF and FGF basic
  27. &nbsp;

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

    Plate 2.0 x 10<sup>6</sup> viable cells in 10 mL of Completed NSC Base Media containing EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated plate
  31. Plate 2.0 x 106 viable cells in 10 mL of Completed NSC Base Media containing EGF and FGF basic onto a Poly-L-ornithine/Fibronectin-coated plate
  32. Incubate the cells at 37 °C and 5% CO2.
  33. &nbsp;

    Replace the media with fresh Completed NSC Base Media every second day
  34. Replace the media with fresh Completed NSC Base Media every second day
  35. Supplement the media with EGF and FGF basic daily
  36. Passage the cells after 3 days or when the cells reach 70-80% confluency.
  37. &nbsp;

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    Verified Customer | Posted 05/13/2021
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FAQs

  • How many times can Mouse Cortical Stem Cells (Catalog # NSC002) be passaged before they lose their multipotency?

    The Mouse Cortical Stem Cells are designated P1 cells as the supplied cells are passaged once. They can be expanded at least three more passages using the monolayer system or at least four more passages using the neurosphere system before their multipotency may be compromised.

  • What is the gender of the mice from which Mouse Cortical Stem Cells are derived?

    Mouse cortical Stem cells are deived by harvesting brain tissue from embryos of pregnant females.Cortical Stem cells from each litter is combined giving a mixture of male and female cells.

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