CELL FREEZING PROTOCOL*

  1)        Replace cell media with fresh media 24-48 hours before freezing.

  2)        Check cells under microscope to make they are healthy before proceeding.

  3)        For optimal results, the cells should be in the mid to late log phase of growth (at or near confluence).

  4)        Aspirate old media, wash with PBS, and aspirate.

  5)        Add 3 to 4mL of trypsin to cells and incubate 3-5 minutes. Cells should appear rounded and will

            begin to come up off the dish.

  6)        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.

  7)        Add media (use volume equal to volume of trypsin used) containing 20% FBS to deactivate trypsin,

            triturate thoroughly.

  8)        Make freezing solution: in 50mL conical tube

                    a. Add 9mL of 20% FBS in normal growth media (DMEM + 1% pen-strep)

                    b. Add 600uL of glycerol (cell culture grade)

                    c. Add 400uL of DMSO

                    d. Vortex mixture

  9)        Transfer cell suspension to 15mL conical tube, centrifuge at 2500 x g x 5 minutes to pellet cells,

            aspirate liquid.

10)        Use 2-3 mLs of freezing solution to resuspend cells, then transfer to remaining freezing solution in

            50 mL conical tube. Gently triturate cells with mixture.

11)        Transfer 1.8mL of cell/freezing solution suspension to 2mL cryogenic vials. 

12)        Place vials in a Styrofoam container and wrap in insulation material (ie surgery pad).

13)        Place vials in a -20 degrees C freezer for one hour.

14)        Transfer vials to a -80 degrees C freezer overnight.  Cells can stay in this freezer for up to two weeks.

15)        Vials can then be transferred to liquid nitrogen for long term storage.

         * Volumes are based on cells grown in a T-75 flask, and can be scaled accordingly. 

sterile conditions.

                     Supplies        Reagent          Vendor                         catalog #

                                          DMEM         Invitrogen                        11965

                                          FBS             Atlanta Biologicals          S11550H

                                          Pen/Strep     Invitrogen                       15140-122

                                          PBS             Invitrogen                       10010

                                          Trypsin         Invitrogen                        25300

                                          Glycerol        Sigma                           G2025

                                          DMSO          Sigma                            D2438