Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.
- Remove and discard culture medium.
Rinse the cell layer with DPBS solution for 2 minutes to remove all traces of serum that contains trypsin inhibitor.
Add 5.0 to 7.0 mL of Trypsin-EDTA solution to the flask and incubate at 37°C .Observe cells under an inverted microscope until cell layer is dispersed (usually within 4 to 6 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
Add 5.0 to 7.0 mL of Trypsin Neutralizing Solution (ATCC® PCS-999-004™) Centrifuge at 125 x g; 10 ± 2 minutes. Discard supernatant and resuspend the cell pellet with 8 mL of complete growth media. Gently break cell pellet by pipetting repeatedly.
Count cells. Seed 2,500 to 5,000 viable cells per cm2. Add appropriate volume of the cell suspension to new culture vessels.
- Incubate cultures at 37°C.
Change media every 2-3 days
Subculture when cells reach 75-80% confluence. Seeding density should be 2,500 to 5,000 viable cells per cm2