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Passage of adherent cell lines (subculture)

Adapted from Freshney, R.I., 1994. Culture of Animal Cells: a Manual of Basic Technique. 3rd Ed.Wiley-Liss. New York. usa

Overview

Remove medium from culture container, rinse with PBS, expose cells to trypsin, incubate, stop trypsin activity by adding serum containing medium, dissociate cells, dilute and return to incubator.

1.

Pipette off spent medium and discard.
2. Add PBS (10 mL/75 cm2 flask) to the flask, being careful not to disturb the monolayer. Rinse the monolayer by gently rocking the flask back and forth. Remove the PBS and discard.
3. Add trypsin (3 mL/75 cm2 flask) and rock the flask to ensure that the entire monolayer is covered with the trypsin solution.
4. Incubate until the cells begin to detach, usually 3–5 minutes. Care should be taken to avoid leaving cells exposed to the trypsin longer than necessary. Care should also be taken that the cells not be forced to detach prematurely, as this may result in clumping.
5.

Add serum-containing medium (3–10 mL/ 75 cm2 flask) and pipette the cells up and down until the cells are dispersed into a single cell suspension.
6.

Count the cells either by hemocytometer or Coulter counter and dilute to the appropriate concentration for seeding.
7.

Add the appropriate volume of cell suspension to a new flask containing medium.
8.

Place flask in incubator, loosen the cap 1/4 turn to allow for gas exchange.

 

 

Discussion

Serum contains trypsin inhibitors. Thus it is important to remove traces of serum in step 2. The serum in the medium in step 5 will inactivate the remaining trypsin and prevent cell damage. If dealing with a serum-free medium, it may be necessary to use an alternative trypsin inhibitor such as soybean trypsin inhibitor. The incubation time for the trypsin and the degree of force required to get the cells into single cell suspension varies between cell lines. It may be necessary to adjust these parameters to suit your particular cells.

 

 

 

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