Complete Growth Medium
The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and place at 37°C to facilitate dispersal. Observe cells within 5 to 10 minutes under an inverted microscope
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- To remove trypsin-EDTA solution, transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
- Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
Recommended seeding densities of 5 x 103 to 4 x 104 viable cells/cm2
- Place culture vessels in incubators at 37°C.
Note: subcultures can also be prepared by scraping the cells from the floor of the flask. Add fresh medium, aspirate the scraped cells to disperse them and dispense into new flasks.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:3 is recommended by depositor
Medium Renewal: 2 to 3 times per week
Freeze medium: Culture medium ,40% ; DMSO,10% ; FBS, 50% FBS..
Storage temperature: liquid nitrogen vapor phase