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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 

1. Remove culture medium with floating cells to a centrifuge tube. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
2. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 10 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
4. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube with the medium and cells from step #1 and spin at approximately 125 x g for 5 to 10 minutes. 
5. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. 6. Place culture vessels in incubator at 37°C.

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Faust JB, Meeker TC. Amplification and expression of the bcl-1 gene in human solid tumor cell lines. Cancer Res. 52: 2460-2463, 1992. PubMed: 1568216

Rieder G, et al. Role of adherence in Interleukin-8 induction in Helicobacter pylori-associated gastritis. Infect. Immun. 65: 3622-3630, 1997. PubMed: 9284128

Mycoplasma contamination was detected in cultures submitted to the ATCC and was eliminated in 1983.