Place flask in an incubator overnight and then observe cells under an inverted microscope. Please contact ECACC within 24 hours if there appears to be a problem. 

Please Note:  Human Cell Bank suspension cell lines should be kept upright and not laid down.

Adherent Cell Lines 

1. Decant most of the medium leaving sufficient to cover the cells (5-8 ml for a 25cm² flask). Gas the flask with 5% CO₂ where appropriate and incubate overnight, at the temperature recommended on the cell line data sheet.
2. At 70-80% confluency decant culture medium and wash cells with PBS. Add 2ml of trypsin/EDTA solution to a 25cm² flask ensuring that all cells are covered. Decant excess trypsin.
3. Incubate at 37^o C (unless otherwise specified on data sheet) until cell sheet detaches, usually 5-10 minutes.
4. Resuspend cells in fresh media at the recommended seeding density (see cell line data sheet).

Note. Some Cell Lines are damaged by tryspin and instead need to be removed by scraping. Please check data sheet. Also, if cells detatched in transit, centrifuge, resuspend pellet and incubate overnight. Cells may re-attach.


Suspension Cell Lines

1. Decant cells into a centrifuge tube and centrifuge at 70-100g for 5 min. Resuspend cells in a 1:1 mixture of fresh and current media at the recommended seeding density. (see cell line data sheet)
   
   Alternatively if cells are growing well, dilute cells with fresh medium. 
   
   Note: Cells that normally form clusters should be diluted rather than centrifuged to minimise cell damage.