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

   the dish.

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.

10) After initial seeding, the media is usually changed every 3-4 days, by aspirating off old media and replacing with

     fresh media. 

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

                                             (40 um)