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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/2.1/2
TITLE: SUBCULTURING ATTACHED AND SUSPENSION CELLS
- The cell line
- Appropriate tissue culture medium (pre-warmed to 37°C) and tested for microbial contaminants
- Centrifuge tubes and Bench top centrifuge
- Tissue culture flasks
- Trypan blue stain (0.4%)
- Ca2+, Mg2+- free PBS for attached cells (pre-warmed to 37°C)
- 0.25% Trypsin/EDTA for attached cells (pre-warmed to 37°C)
- Inverted microscope
For Attached Cell Lines
Where specific seeding densities are required centrifuge cell suspension at 200g for 5 minutes and discard supernatant.
Resuspend the cells or cell pellet at required seeding density in fresh media in new flasks or roller bottles.
- Check the cells for microbial contamination by microscopic examination.
- Check cells for attachment, then decant medium and wash the cell layer twice with PBS (i.e. approximately 1ml per 10cm2 surface area).
- Add trypsin/EDTA (i.e. approximately 0,5 to 2ml per 25cm2 surface area). Spread the trypsin over cell layer.
- Incubate at 37 ± 1°C until the cells are detached (approx. 5 to 10 min). Once cells start to detach, tap flask gently and check that they are all loose using microscope.
- Wash cells into corner of flask with fresh medium (2 to 3ml/25cm2).
- Pipette gently to break-up the clumps.
- Perform a viable cell count where necessary, e.g. when preparing cells for cryopreservation, by taking up a sample and counting by the trypan blue exclusion method (
For Suspension Lines
Follow the protocol above except omit steps 2 to 6.
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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