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.
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.
