eChapter Name: Culture and Sub-culturing of Continuous Cell Lines
9789358877724
eBook Name: ANIMAL CELL CULTURE
by Ratan Kumar Choudhary, Manu M, Yashpal Singh MaliK
What is subculture?
In cell culture, a process called subculturing, or passaging, keeps cells alive and growing. It involves taking cells from an existing container, removing the used medium, and placing them in a new container with fresh nutrients. This allows the cells to continue multiplying and creates more cells for research.
Adherent subculture protocol Cells nearing 80% coverage need splitting!
1. Warm things up: Get your media, detachment solution, and incubator ready at 98.6°F (37°C).
2. Safety first! Disinfect your work area and supplies inside a biosafety cabinet.
3. Gently rinse: Remove the old media and wash the cells with a salt solution at room temperature. Be careful not to dislodge the cells.
4. Detach the cells: Add a pre-warmed enzyme solution to detach the cells from the flask surface. Keep an eye on them to make sure it works (usually 2 minutes). Note: Not all cells need this enzyme, and it can be harmful to some.
5. Stop the enzyme: Once all the cells are detached, add a culture medium with serum to neutralize the enzyme.
6. Collect the cells: Transfer the cell suspension to a tube, rinse the flask with fresh medium to get all the cells, and add it to the same tube.
7. Spin it down: Centrifuge the cell suspension for 5 minutes at room temperature (1000 rpm).
8. Get ready for counting: Remove the liquid, loosen the cell clump, and resuspend the cells in a fresh medium at a good volume for counting.
9. Count your cells: Use a hemocytometer to count the cells and determine their health.