- 1x Phosphate-buffered saline (PBS)
- Trypsin-EDTA buffer/ media
- Complete cell culture medium
- 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.
7.) 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. 8.) If the cells are not fully detached, place the flask back into the incubator. Some cells may require some mechanical agitation (including “rapping” the flask or “scraping” the culture surface), BUT THIS IS NOT PREFERRED. 9.) Once the cells have detached, add serum-containing medium to the flask in an amount approximately 2-3X that of the trypsin (i.e., 10-15ml of medium for a T-75 culture flask). Trypsin will start to act on the excess serum proteins instead of harming the cells. Note: The medium MUST contain serum in order to act to inhibit the trypsin. Serum-free media can only be used IF a trypsin inhibitor is used. 10.) Collect the harvested cells and pipet into an appropriately sized centrifuge tube. 11.) Centrifuge cells for approximately 5 minutes at 200xg (800-1100 rpm, depending on the centrifuge). 12.) During centrifugation, label new culture flasks. Label flasks with cell type, your initials, the new passage number (passage number increases with every split), and today’s date. 13.) NOTE: Certain cell types are only viable up to a certain passage number, such as primary cells. Make sure to check this before splitting beyond the appropriate passage number! 14.) Following centrifugation, aspirate the media above the cell pellet and resuspend the cells in a logical volume (5-10 ml). 15.) If necessary, count cells via hemacytometer or coulter counter.16.) Resuspend your pelleted cells in an appropriate volume of growth medium and dispense into sterile flasks or onto your experimental surfaces.