1. Thaw cell culture freezing medium completely and mix well by gently swirling bottle. Keep freezing medium on ice during use.

2. Cells to be frozen should be in late log phase growth.

3. Monolayers will need to be dissociated. After dissociation, cells are resuspended in Neural Stem Cell Basal Medium (Cat. No. SCM003) and counted to determine viability and number.

4. Centrifuge cells at 300 x g for 3 min. Remove the medium above the pellet.

5. Resuspend the cells in Neural Stem Cell Freezing Medium at a concentration of ~4 x 10⁶ cells/mL. Freeze 1 mL of cells/vial. After the cells have been resuspended and aliquoted into appropriate cryogenic storage vials, they can be placed in a freezing container and the normal freeze down procedure should be started within five minutes.

6. Cells must be stored at or below -80°C. For long term storage the cells should be stored in ultra-low temperature freezer (-150°C), or in liquid nitrogen (-196°C).

7. Thawing of cryopreserved cells should be as follows:

a. Do not thaw the cells until the recommended medium and appropriately coated poly-L-ornithine and laminin plasticware and/or glassware are on hand.
b. Thaw cells quickly in a 37°C water bath. Important: Do not vortex the cells.
c. Sterilize vial by rinsing with 70% ethanol.
d. In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical tube. Be careful to not introduce any bubbles during the transfer process.
e. Using a 10 mL pipette, slowly add dropwise 9 mL of Neural Stem Cell Basal Medium (pre-warmed at 37°C) to the 15 mL conical tube. IMPORTANT: Do not add the whole volume of medium at once to the cells. This may result in decreased cell viability due to osmotic shock.
f. Gently mix the cell suspension by slow pipeting up and down twice. Be careful to not introduce any bubbles.

IMPORTANT: Do not vortex the cells.
g. Centrifuge the tube at 300 x g for 2-3 minutes to pellet the cells.
h. Decant as much of the supernatant as possible
i. Resuspend the cells in a total volume of 10 mL of Neural Stem Cell Basal Medium (pre-warmed at 37°C) containing freshly added growth factors.

Note: Growth factors should always be added fresh to the Neural Stem Cell Basal Medium. For rat neural stem cells: add 2-4 μL of FGF-2 (100 μg/mL) stock to 10 mL of Neural Stem Cell Basal Medium. For mouse neural stem cells, add 2 μL of FGF (100 μg/mL stock), EGF (100 μg/mL stock) and heparin (10 mg/mL stock) to 10 mL of Neural Stem Cell Basal Medium.

j. Plate the cell mixture onto a poly-L-ornithine and laminin-coated 10-cm tissue culture plate.
k. Incubate the cells at 37°C in a 5% CO2 humidified incubator.
l. The next day, exchange the medium with fresh Neural Stem Cell Basal Medium (pre-warmed to 37°C) containing freshly added growth factors (refer to step 7i for growth factor concentrations). Exchange with fresh medium containing growth factors every other day thereafter.
m. When the cells are approximately 80% confluent, they can be dissociated with TM=["Accutase"] (Cat. No. SCR005) and passaged or alternatively frozen for later use.

Serum-free formulation. Contains 10% DMSO.

Store at -20°C.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.