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

Catalog # NSC001 | R&D Systems, Inc. a Bio-Techne Brand

Key Product Details

Rat Cortical Stem Cells (3 x 10^6 cells/vial) (Catalog # NSC001)
(4)
Rat Cortical Stem Cell Multipotency Following Expansion
Rat Cortical Stem Cells Express Nestin and SOX2
Differentiation of Rat Cortical Stem Cells
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Setting the standard in quality research reagents

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

Neurosphere Expansion

Perform a cell count
  • Perform a cell count.
 

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
  • 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.
&nbsp;

Replace the media every 4 days according to the number of neurospheres present
  • 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.
  • Passage the cells at 5-6 days or when the neurospheres have a dark clump inside or ruffling on the outside.
&nbsp;

Passing Neurosphere

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

Partially dissociate the neurospheres by pipetting up and down 20 times with a P200 pipette
  • 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.
&nbsp;
  1. Add 1 mL of fresh pre-warmed media to the vial of frozen rat cortical stem cells.
  2. See&nbsp;Details

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.
&nbsp;
  • 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.
  • 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.
&nbsp;

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

Replace the media once cells become adherent. After 24 hours, add 10 µL of FGF basic stock (1000X) to the culture
  • Replace the media once cells become adherent. After 24 hours, add 10 &mu;L 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.
&nbsp;

Passaging Cells

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

Scrape the cells from the plate
  • 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.
&nbsp;

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

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

Replace the media with fresh Completed NSC Base Media every second day
  • 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.
&nbsp;
  1. Coat cell culture plates with Poly-L-ornithine and Fibronectin.
  2. See&nbsp;Details

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    Name: Anonymous
    Verified Customer | Posted 12/01/2017
    These cells have grown well and have been used for differentiation studies.
    Rat Cortical Stem Cells (3 x 10e6 cells/vial) NSC001

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

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