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Summary Passaging Cell Culture Guide
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A detailed explanation of how to split cell cultures and why each step is taken. Created by a graduate Biochemistry student who is now studying Biological Sciences at Cambridge University. This is designed to be used for all levels of learning, from GCSE to university. When learning biology at scho...

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  • August 6, 2024
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  • 2024/2025
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Passaging Cells
General Protocol for splitting cell culture


PREPARATION
1 Begin by sterilising the work area and all
equipment in the cell culture hood to maintain a
sterile environment and prevent contamination.

Warm the complete growth medium and trypsin-
EDTA (or trypsin-versene) to 37ºC. This step is
crucial as it prevents temperature shock to the
cells and ensures that trypsin functions effectively


CONFLUENCY
2 Using a microscope, check the confluency of your cell
culture. Cells are typically ready to be passaged when
they reach 70-80% confluency. Overconfluency can
lead to increased cell death due to competition for
nutrients and space.




WASHING
3 Remove the existing culture media from the flask and
wash the cells with a sufficient amount of phosphate-
buffered saline (PBS). Gently rock the flask to ensure
even washing. This step helps to remove debris and dead
cells, which can negatively impact cell health and
experimental results.



DETACH
4 Add the required amount of trypsin-
EDTA/versene to the culture flask. Trypsin is an
enzyme that detaches adherent cells from the
flask surface. Incubate the flask for 5 minutes at
37ºC. After incubation, gently the tap the side of
the flask to dislodge the cells and check under the
microscope to ensure they are in a single-cell
suspension.

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