Passaging Cells: Cell Culture Basics
Thermo Fisher Scientific・2 minutes read
Cell growth in culture follows a standard pattern, and passaging is necessary to maintain healthy cell numbers for experiments. To ensure successful cell handling, sterilize the hood, gather supplies, examine cultures for contamination, rinse cells, confirm cell release, count cells accurately, differentiate live and dead cells, and transfer to fresh flasks for optimal seeding density.
Insights
- Cell growth in culture progresses through distinct phases, necessitating passaging to maintain healthy cell numbers for experiments once cells reach confluence or exceed the medium's capacity.
- Prior to handling cells, it is crucial to sterilize the environment, inspect cultures for contamination, utilize appropriate solutions for rinsing and detachment, confirm cell release and viability, and ensure proper seeding density in fresh flasks for optimal growth conditions.
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Recent questions
How does cell growth progress in culture?
Cell growth in culture begins with a lag phase after seeding, followed by the log phase of exponential growth. Passaging becomes necessary when cells cover the plate or exceed the medium's capacity.
What steps are essential for successful cell handling?
To ensure successful cell handling, sterilize the hood, gather necessary supplies, examine cultures for contamination, rinse adherent cells with a balanced salt solution, and use a dissociation reagent like TrypLE Express.
How can cell release be confirmed after dissociation?
After dissociation, confirm cell release using a microscope and ensure a single-cell suspension for accurate cell counting. Differentiate live and dead cells with Trypan blue before resuspending in warm complete growth medium.
What is the optimal seeding density for cell transfer?
Resuspend cells in warm complete growth medium before transferring to fresh flasks for optimal seeding density. Cap flasks tightly for gas exchange and evenly distribute cells before returning to the incubator.
Why is passaging necessary in cell culture?
Passaging is necessary when cells cover the plate or exceed the medium's capacity to maintain healthy cell numbers for experiments. It helps prevent overcrowding and ensures the continued growth and health of the cell culture.
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Summary
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Optimizing Cell Culture Handling for Experiments
- Cell growth in culture follows a standard pattern, starting with a leg after seeding and moving into the log phase of exponential growth. Passaging is necessary when cells cover the plate or exceed the medium's capacity to maintain healthy cell numbers for experiments.
- To ensure successful cell handling, sterilize the hood and gather all necessary supplies before retrieving flasks from the incubator. Carefully examine cultures for contamination or deterioration. For adherent cells, use a balanced salt solution like DPPS to rinse the cells, followed by a gentle dissociation reagent such as TrypLE Express to detach cells from the plate.
- After dissociation, confirm cell release using a microscope and ensure a single-cell suspension for accurate cell counting. Use Trypan blue to differentiate live and dead cells, then resuspend the cells in warm complete growth medium before transferring to fresh flasks for optimal seeding density. Cap flasks tightly for gas exchange and evenly distribute cells before returning to the incubator.
