Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.
Reagents and Equipment required:
- Complete cell culture media
- Cell culture vessels (48-well, 24 well, 12 well and 6 well cell culture plates; cell culture flasks (T25, T75 T175)
- TrypLE® or equivalent cell dissociation buffer (Life Technologies 12605010)
- Phosphate buffered saline
- Mark the well of positive clones for expansion from the screening agarose-gel image.
- Pre warm the appropriate culture media for the cell line in a 37°C water bath.
- Obtain the 96-well plate and mark the position of the clone on the lid for easy identification. Remove the culture media to waste.
- Wash the cells with 100 μL of 1xPBS and then add 50 μL of TrypLE®.
- Incubate plate in a 37°C incubator for 5 minutes (or until cells have detached).
- Add 100 μL of pre-warmed culture media to the TrypLE® treated cell suspension and transfer to a well of a 48-well plate containing approximately 350 μL of culture media.
- Add 200 μL of culture media back to the original 96-well.
- Incubate both plates in a humidified incubator under appropriate conditions (e.g., 37°C, 5% CO2 atmosphere) until the 48-well of cells reach confluence.
- Repeat the expansion process, scaling the reagents for the increasing vessel size and expand the cells to a minimal density for a 10-15 vial bank of cells at 1x106/vial e.g. a T75-T175 as appropriate.