Complete Growth Medium
NeuroCult NS-A Basal Medium (StemCell Technologies #05750) with NS-A Proliferation Supplement (StemCell Technologies #05754) + 20 ng/mL EGF (StemCell Technologies #78003.1) + 20 ng/mL bFGF (Peprotech #AF-100-15) + 2 µg/mL Heparin (StemCell Technologies #07980).
Prepare media according to the manufacturer’s instructions: Stem Cell Technologies Catalog #5751
Culture vessels must be pre-coated with laminin prior to seeding with cells.
Coating procedure (perform all steps in a biosafety cabinet):
- Thaw (Corning #354232) laminin on ice.
- Transfer 10 mL of D-PBS (ATCC 30-2200) to a 15 mL conical tube.
- Add 100 µL stock laminin to the 15 mL conical tube with D-PBS and mix thoroughly.
- Added diluted laminin to vessels at a volume of approximately 1 mL per 10 cm2. For example, use 7.5 mL diluted laminin for a 75 cm2 flask.
- Tilt plate to ensure entire surface is coated.
- Place in a humid cell culture incubator at 37oC for 1-24 hours.
Immediately prior to seeding cells, aspirate off the laminin coating solution and discard. Do not allow the plates to dry out.
- Passage cells when the culture has reached approximately 70% to 80% confluence.
- Pre-coat new culture vessels with laminin, if necessary.
- Warm Accutase and complete growth media to room temperature.
- For each flask, carefully aspirate the spent media without disturbing the monolayer.
- Rinse the cell layer one time with 3 to 5 mL D-PBS (ATCC 30-2200) to remove residual medium.
- Add room temperature Accutase (1 to 2 mL for every 25 cm2) to each flask.
- Gently rock each flask to ensure complete coverage of the Accutase solution over the cells, and then aspirate the excess fluid off of the monolayer.
- Observe the cells under the microscope.
- When the majority of cells appear to have detached (typically 2-5 minutes), quickly add an equal volume of the complete growth medium to each flask.
- Transfer the dissociated cells to a sterile centrifuge tube.
- Centrifuge the cells at 200 x g for 5 minutes.
- Aspirate neutralized dissociation solution from the cell pellet and resuspend the cells in 2 to 8 mL fresh, pre-warmed, complete growth medium.
- Count the cells and seed new culture flasks at a density of 5 x 104 viable cells per cm2. Prior to seeding, aspirate the coating and discard the coating laminin solution from the vessel.
- Place newly seeded flasks in a 37°C, 5% CO2 incubator for at least 24 to 48 hours before processing the cells further.
- Perform a complete medium change every 3-4 days.
Complete growth media containing 10% DMSO (ATCC 4-X).
Atmosphere: air, 95%; carbon dioxide (CO2), 5%