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

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

View Graphic
Procedure

View Graphic
Procedure

 

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

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

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

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

Thawing Cryopreserved Mouse Cortical Stem Cells

• 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.
• Centrifuge the cells at 200 x g for 5 minutes.

 

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• Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing EGF and FGF basic.

 

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Neurosphere Expansion

• Perform a cell count.

 

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• 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.
• Incubate the cells at 37 °C and 5% CO₂.
• Add fresh EGF and FGF basic daily to the media.

 

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• 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.
• Passage the cells at 5-7 days or when the neurospheres have a dark clump inside or ruffling on the outside.

 

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Passing Neurosphere

• Transfer the media containing the floating neurospheres to a 15 mL tube.
• Centrifuge for 5 minutes at 100 x g.

 

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• Partially dissociate the neurospheres by pipetting up and down 20 times with a P200 pipette.
• At passages 1 and 2 the cells should be split 1:1. After passage 2 the cells can be split 1:2.

 

1. Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells
2. See Details

Thawing Cryopreserved Mouse Cortical Stem Cells

• Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells.
• 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.

 

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

 

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Monolayer Expansion

• 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.
• Incubate the cells at 37 °C and 5% CO₂.

 

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• 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.
• Replace the media with fresh Completed NSC Base Media every second day.
• Supplement the media daily with EGF and FGF basic
• Passage the cells when they reach 70-80% confluency.

 

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Passaging Cells

• Wash the cells once with pre-warmed HBSS.
• Add 5 mL of HBSS.
• Incubate at room temperature until the cells round up.

 

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• Scrape the cells from the plate.
• Transfer the cells to a 15 mL centrifuge tube.
• Centrifuge for 5 minutes at 200 x g.
• Remove the supernatant.

 

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• Resuspend the cells in 5 mL of Completed NSC Base Media containing EGF and FGF basic

 

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

 

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• 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
• Incubate the cells at 37 °C and 5% CO₂.

 

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

 

1. Coat cell culture plates with Poly-L-ornithine and Fibronectin.
2. See Details

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