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Culture Collections

How to handle growing cells on receipt

 

The transport medium does not include antibiotics. Immediately check the cells upon arrival for any obvious defects by using an inverted microscope and then lay the flask flat in an incubator, set at the temperature specified in the cell line data sheet, overnight. If the cell density is too high then follow the procedure below and as described in the cell line data sheet. Please contact us within 24 hours if there appears to be a problem.

 

Adherent Cell Lines

1. Following overnight incubation, remove most of the transport medium leaving sufficient medium to cover the cells (5-8ml for a 25cm2 flask). Incubate at the temperature and CO2 level recommended on the cell line data sheet until the required degree of cell confluence is achieved.

2. At 80% confluence, unless specified otherwise on the cell line data sheet, carefully remove the culture medium and wash the monolayer of cells twice with PBS. Add 1-2ml of 10.25% Trypsin/ 0.2% EDTA solution to the 25cm2 flask ensuring that all cells are covered. Decant the excess Trypsin/EDTA immediately.

3. Incubate (unless specified otherwise on data sheet) until the cells start detaching from the flask – usually takes 2-10 minutes.

4. Add 5ml fresh pre-warmed (to incubation temperature specified on data sheet) medium to resuspend the cells and quench the trypsin. Determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells. Seed into a fresh flask(s) adjusting the volume of the medium, and if necessary the flask size, to achieve the cell seeding density recommended on the cell line data sheet.

1Note: Trypsin can cause damage to some cell lines. Before commencing any work, please check the cell line data sheet to determine if the use of trypsin is appropriate and for specific procedures when using serum free media.

 

 

Suspension Cell Lines

1. Following overnight incubation, determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells. Seed at the cell seeding density recommended in the data sheet by adjusting the volume using fresh pre-warmed medium in a larger flask if necessary.

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