SEEDING AND PASSAGING CELLS
1) Heat sterile media (DMEM, 10%FBS, 1%Pen/Strep) and Trypsin to 37 degrees C.
Under a sterile hood with sterile technique:
2) Aspirate media from flask using sterile glass pipets. Take care not to disturb cells or cross contaminate flasks.
Add 5 mLs of sterile PBS to each flask to rinse residual media. Aspirate PBS.
3) Add 3-4mL of trypsin to cells and incubate 3-5 minutes. Cells should appear rounded and will begin to come up off
4) Triturate trypsin to get cells into suspension. Check flask under microscope to be sure that all cells are in
suspension. If cells are still attached wait an additional 5 minutes and triturate again. If cells continue to adhere,
add an additional 3-4 mL of trypsin, wait 5 minutes, and repeat trituration step.
5) Add media (use volume equal to volume of trypsin used) containing 20% FBS to deactivate trypsin, triturate
thoroughly to wash the cells from the flask and to break up clumps of cells.
6) Using a cell strainer, transfer suspension to a 50 mL conical vial. Wash strainer with an additional 2-3 mL media.
7) Take a 10 uL sample to obtain cell density from the Countess. Alternatively a hemocytometer may be used
to determine cell counts/volume.
8) Transfer volume of media required to seed cells into appropriate container (eg sterile 50 mL conical tube). Add
desired number of cells (from cell count determined by Coulter counter/hemocytometer) and triturate cells with
media. Transfer desired volume of cell suspension to culture or experimental dishes.
9) Excess cells may be re-seeded into a new flask or cell culture dish. Add extra media, if needed, appropriate for size
of dish used. Be sure to indicate on the flask/dish the cell type, the date, and the new passage number. Every time
cells are passaged (trypsinized), the passage number is increased by 1.
We use 15 mL total volume of media for a T-75 flask
10) After initial seeding, the media is usually changed every 3-4 days, by aspirating off old media and replacing with
11) Allow cells sufficient time to recover from trysinization, at least 7 days, before splitting and reseeding. We
prefer that cells are allowed to reach confluence before they are trypsinized again.
Vendor catalog #
Reagents DMEM Invitrogen 11965
FBS Atlanta Biologicals S11550H
Pen/Strep Invitrogen 15140-122
PBS Invitrogen 10010
Trypsin Invitrogen 25300
Supplies Cell Strainer BD Falcon 352340
This protocol is used for all of the rat and mouse cells we culture.