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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/4/2.2 Appendix 4


TITLE: HELPFUL HINTS - ECACC -


PROCEDURES FOR HANDLING CELL LINES UPON RECEIPT

FROZEN CELLS

Transfer ampoule(s) to a storage vessel if it is not to be used immediately. Cells should be stored in the gaseous phase of liquid nitrogen if not to be used immediately.

Note: This may result in some loss of viability.

GROWING CELLS

On arrival check the cells under an inverted microscope and incubate overnight.

Please contact ECACC within 24 hours if there appears to be a problem.

SUBCULTURING CELLS

A. ADHERENT CELLS

  1. Upon receipt of a growing culture of an attached cell line decant most of the medium leaving sufficient to cover the cells; 5-8ml for a 25cm flask. Re-gas with 5% CO2 where appropriate and incubate overnight at temperature recommended on the cell line data sheet.
  2. When the cells reach confluence they should be split into new flasks at a ratio recommended on the data sheet supplied with each individual cell line.
  3. In order to split the cells they should be detached from the flask by trypsinisation as follows:

    i) wash the cells twice with PBS

    ii) add the appropriate amount of trypsin/EDTA solution (1ml per 25cm), swirl to ensure all the surface is covered, then decant most of the solution leaving just sufficient to wet the cells.

  4. Incubate the flask at 37C for 5-10 minutes or until the cell sheet detaches.
  5. the cells are finally resuspended in the appropriate medium.

B. SUSPENSION CELL LINES

  1. Upon receipt of a growing culture of a suspension cell line decant cells into a centrifuge tube and centrifuge for five minutes at 70-100g. Resuspend the cells in a 1:1 mixture of fresh and curernt medium in recommended seeding density.
  2. When cells reach their maximum cell density they should be resuspended in fresh medium at between 2-500 cells/ml.

Note:

- CELLS THAT GROW AS CLUSTERS: cells that normally form clusters should be allowed to settle, the exhausted medium pipetted off and the cells resuspended in fresh medium as centrifugation may cause n cell damage.

- SEMI-ADHERENT CELL LINES : certain cell lines grow in suspension and also attach to the tissue culture flask. Knock the flask gently to dislodge the cells and treat as a suspension cell line.


Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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