Subculturing
Volumes are given for a 75 cm
2 flask. Increase or decrease the amount of dissociation medium needed proportionally 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.
-
Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
-
Add 6.0 to 8.0 mL of RPMI 1640 medium supplemented with 5% fetal bovine serum, aspirate cells by gently pipetting and transfer to a centrifuge tube. Spin at 125 x g for 5 to 10 minutes. Discard supernatant.
- Resuspend the pellet in RPMI 1640 medium and dispense into new flasks.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: Add fresh medium twice weekly
