Rat Cortical Stem Cells (3 x 10e6 cells/vial)
R&D Systems, part of Bio-Techne | Catalog # NSC001
Key Product Details
|
Verification of Neural Progenitor Cell Multipotency Following Expansion with N-2 MAX Media Supplement. Rat Cortical Stem Cells were differentiated for 7 days in media supplemented with N-2 MAX Media Supplement (Catalog # AR009). Differentiated cells were stained with a Mouse Neuron-specific Anti-beta-III Tubulin Monoclonal Antibody (Catalog # MAB1195) followed by the NorthernLights™ (NL)557-conjugated Secondary Antibody (Catalog # NL007; red) to detect neurons. The cells were stained with a Sheep Anti-Human/Rat GFAP Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2594) followed by the NL557-conjugated Secondary Antibody (red; Catalog # NL010) to detect astrocytes; and cells were stained with a Mouse Anti-Human/Mouse/Rat/Chicken Oligodendrocyte Marker O4 Monoclonal Antibody (Catalog # MAB1326), followed by the NL557-conjugated Secondary Antibody (Catalog # NL019) to detect oligodendrocytes. The nuclei were counterstained with DAPI (blue). |
|
Neural Progenitor Cells Expanded with N-2 Plus Media Supplement Express Nestin and SOX2. Rat Cortical Stem Cells (Catalog # NSC001) were cultured for 7 days in media supplemented with 1X N-2 Plus Media Supplement (Catalog # AR003) and 20 ng/mL of Recombinant Human FGF basic (Catalog # 233-FB). The cells were stained with a PE-conjugated Mouse Anti-Human Nestin Monoclonal Antibody (Catalog # IC1259P, red histogram), a PE-conjugated Mouse Anti-Human/Mouse SOX2 Monoclonal Antibody (Catalog # IC2018P, green histogram), or a PE-conjugated Mouse IgG2A Isotype Control Antibody (Catalog # IC003P, open histogram). Under these conditions, cells were shown to express high levels of neural multipotency markers Nestin and SOX2. |
|
Differentiation of Rat Cortical Stem Cells. Neural progenitors were cultured for seven days in DMEM/F12 containing N-2 Plus Media Supplement (Catalog # AR003) without FGF Basic to induce differentiation. The differentiated cells were labeled with a Goat Anti-Rat Nestin Affinity-purified Polyclonal Antibody (Catalog # AF2736) followed by the NorthernLights™(NL)493-conjugated Donkey Anti-Goat IgG Secondary Antibody (Catalog # NL003; green) and a Mouse Neuron-specific Anti-beta-III Tubulin Monoclonal Antibody (Catalog # MAB1195) followed by the NL-557-conjugated Donkey IgG Anti-Mouse Secondary Antibody (Catalog # NL007; red). Nuclei were counterstained with DAPI (blue). |
Assay Procedure
Refer to the product datasheet for complete product details.
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
- 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.
- Centrifuge the cells at 200 x g for 5 minutes.
- Resuspend the cell pellet in 10 mL of Completed NSC Base Media containing FGF basic.
Neurosphere Expansion
- Perform a cell count.
- 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.
- Incubate the cells at 37 °C and 5% CO2.
- Add fresh FGF basic to the media daily.
- 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 FGF basic.
- • Add the neurosphere suspension to 5 mL of fresh Completed Base Media containing FGF basic in one well of a 6-well plate.
- a. Less than 50 neurospheres:
- Passage the cells at 5-6 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 passage 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 rat cortical stem cells.
- See Details
Procedure Overview for Expanding Rat 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 rat 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 FGF basic.
- Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
- Perform a cell count.
Monolayer Expansion
- 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.
- 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) to the culture.
- Replace the media with fresh Completed NSC Base Media every second day.
- Supplement the media daily with FGF basic.
- Passage the cells when they reach 60-70% 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 50 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 FGF basic.
- Mix 10 μL of the cell suspension with 10 μL of 0.4% Trypan Blue
- Perform a cell count.
- 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.
- 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 FGF basic daily.
- Passage the cells after 3 days or when the cells reach 70% confluency.
- Coat cell culture plates with Poly-L-ornithine and Fibronectin.
- See Details