- 1x Phosphate-buffered saline (PBS)
- Trypsin-EDTA buffer/ media
- Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
- Pre-warm all reagents to 37°C in the bead bath.
- After reagents are warmed, spray bottles down with ethanol and prepare the hood as for routine work.
- Aspirate old culture media from the cell culture vessel.
- Add PBS to the cells. Lay the flask flat and wash by gently tilting the flask back and forth.
Note: For a T-75 flask, use 5 mL PBS. For a T-25, use 2 mL PBS.
- Aspirate off the PBS.
- Add Trypsin-EDTA into the culture flask. Lay the flask flat and coat the cells completely with the Trypsin-EDTA. Let the flask sit in the hood at room temperature for 5 minutes.
Note: For a T-75 flask, use 2 mL PBS. For a T-25, use 0.5 mL PBS.
- After incubation, examine the cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask/dish.
- If the cells are not fully detached, place the flask back into the 37 °C incubator. Some cells may require some mechanical agitation (including “tapping” the flask).
- Once the cells have detached, add serum-containing medium (e.g., 10% FBS) to the cells to deactivate the trypsin.
Note: For a T-75 flask, use 8 mL medium. For a T-25, use 3.5 mL medium.
- Get a new flask, label it with the cell line name, your initials, and the date. Include the passage number (e.g., PS-1 if you are splitting the cells for the first time, PS-2 for the second time, etc.).
- If you are splitting 1:10, add 9 mL medium to the new flask. In the old flask, gently resuspend the cells by pipetting up and down about 5 times. Transfer 1 mL of cells into the 8 mL of medium in the new flask. Lay the flask flat and tilt back and forth to spread the cells evenly.
Note: For less robust cells,split 1:5 by adding 2 mL cells to 8 mL fresh medium, or 1:4 by adding 2.5 mL cells to 7.5 mL medium.
- Place the new flask into the 37 °C incubator. The cells should attach in a couple of hours.