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[Protocol] Expansion of cell line clones from single cells

Jun 10, 2016 2:23:34 PM No Comments

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
  • Stripettes

Protocol

  1. Mark the well of positive clones for expansion from the screening agarose-gel image.
  2. Pre warm the appropriate culture media for the cell line in a 37°C water bath.
  3. Obtain the 96-well plate and mark the position of the clone on the lid for easy identification. Remove the culture media to waste.
  4. Wash the cells with 100 μL of 1xPBS and then add 50 μL of TrypLE®.
  5. Incubate plate in a 37°C incubator for 5 minutes (or until cells have detached).
  6. 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.
  7. Add 200 μL of culture media back to the original 96-well.
  8. 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.
  9. 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.
Notes
  • Cells can be banked at an earlier time point during the expansion e.g. 6-well stage – this yields fewer frozen vials but ensures an early stage bank of cells that can be revived should problems be encountered during expansion and banking.
  • Point 7 above is essential to propagate a back-up culture of the cell line, which can be used if the initial expansion of the cells to 48-well cell culture plates shows poor recovery.
#Cell lines, #Gene editing, #Protocols

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