Cell passaging refers to the process of dividing cells into smaller portions, reseeding them into another culture container, and then culturing them again. Cell culture flasks are a commonly used consumable during passaging. So what are the specific steps for cell passaging?
1.Take out the cell culture flask from the incubator, spray 75% alcohol on the surface of the flask with an alcohol spray flask, and transfer it to the ultra-clean bench.
2. Open the lid of the cell culture flask, gently aspirate the old medium, add an appropriate amount of PBS buffer solution with a Pasteur pipette, wash it 1-2 times, and discard the PBS buffer solution.
3. The cell culture flask can be supplemented with 1-2ml of trypsin with a pipette, and the "cross" is shaken to fully contact the trypsin and cells.
4. Transfer the cell culture flask containing trypsin to the inverted microscope stage, observe the digestion of cells under the microscope, and when the cells become rounded and a large number fall off, prepare to terminate digestion. Spray 75% alcohol on the surface of the flask and transfer it to the ultra-clean bench.
5. The cell culture flask is supplemented with an equal amount of serum-containing medium, and digestion is terminated. The pipette is fully blown and beaten to make the cells completely fall off.
6. Transfer the mixed liquid in the cell culture flask to a centrifuge tube, balance it, and centrifuge for about 5 minutes.
7. After the cell culture flask is centrifuged, spray the surface of the centrifuge tube with alcohol spray flask, transfer it to the ultra-clean bench, open the lid, and pour the supernatant into the waste liquid cylinder.
8. Add an appropriate volume of serum-containing medium to the cell culture flask, and gently beat it with a pipette or Pasteur pipette to prepare a cell suspension.
9. Divide the cell suspension into 2 (or more) sterile and clean cell culture flasks, add an appropriate amount of medium, and close the lid.
10. Place the divided cell culture flasks on the inverted microscope stage and observe the cell situation. The number of cells should be guaranteed. Too few cells will affect their growth.
11. Make a label on the flask of the cell culture flask, indicating the passage time, cell type, operator, and other information. Before putting it into the incubator, spray some alcohol on the surface of the flask, organize the operation table, wipe the ultra-clean bench with 75% alcohol, and clean up the waste liquid and garbage.
The above is the specific process of cell passaging using cell culture flasks. When operating, attention should be paid to wearing sterile clothing, sterile gloves, and masks, and the entire operation should be done aseptically to avoid contamination of the cells.