Wittrup: Subculturing Protocol

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The following protocol details how to lift cells from adherent monolayers and seed a subculture. Typically, cells are subcultured when they reach 80% confluence, and are commonly split 1:4 to 1:10.


warm media

warm lifting agent (such as trypsin EDTA, versene, etc)

PBS (phosphate buffered saline)


Aspirate off culture media.

Wash cells with 1/2 to 1 volume PBS and aspirate (this is done to remove FBS).

Add 1/4 to 1/2 volume (enough to cover the dish) lifting agent and return to incubator for a few minutes.

Check the progress of the reaction under the microscope periodically. It is unnecessary to wait until all cells are detached, as media can be pipeted over the surface of the dish to detach any remaining adherent cells. A 4 min incubation is often sufficient.

Add a volume of media equal to the volume of lifting agent (this is done to "quench" the trypsinization of the cells) and pipet over the dish several times to dislodge remaining adherent cells.

Place the cell suspension in an appropriately sized tube and centrifuge. 2 minutes at a speed of 1.4 on our centrifuge is likely to do.

Aspirate the supernatant above the cell pellet, and resuspend in the desired volume of media (for example, 10 ml). Once in suspension, cell number can be quantified for experimental work.

Remove the desired amount of the cell suspension to a fresh dish and add media to the desired volume (for example, 2 ml of the 10 ml suspension for a 1:5 split, then add 8 ml fresh media for a total of 10 ml plated out on a 100 mm dish).