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
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
Procedure Overview for Expanding Mouse Cortical Stem Cells Using the
Neurosphere System
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.
- Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing EGF and FGF basic.
Neurosphere Expansion
- 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.
- Replace the media every 4 days according to the number of neurospheres present:
- a. Less than 50 neurospheres:
- • 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.
- b. More than 50 neurospheres:
- • Transfer the media containing the neurospheres to a 15 mL tube.
- • Centrifuge for 5 minutes at 100 x g and remove the media.
- • Resuspend the pellet using a small quantity of fresh Completed NSC Base Media containing EGF and FGF basic.
- • 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.
Passing Neurosphere
- Transfer the media containing the floating neurospheres to a 15 mL tube.
- Centrifuge for 5 minutes at 100 x g.
- 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.
- Add 1 mL of fresh pre-warmed media to the vial of frozen mouse cortical stem cells
- See Details
Procedural Overview for Expanding Mouse Cortical Stem Cells Using the
Monolayer System
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.
- Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
- Perform a cell count.
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.
- 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.
Passaging Cells
- Wash the cells once with pre-warmed HBSS.
- Add 5 mL of HBSS.
- Incubate at room temperature until the cells round up.
- 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.
- Resuspend the cells in 5 mL of Completed NSC Base Media containing EGF and FGF basic
- Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
- Perform a cell count.
- 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.
- 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.
- Coat cell culture plates with Poly-L-ornithine and Fibronectin.
- See Details
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