Love:TCProcedures:Freezing Cells

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This procedure describes a standard method for freezing mammalian cells for long-term storage in liquid nitrogen. The specific example described is for mouse hybridomas, but the principles are the same for other types of cells.

1. Culture hybridoma cells in complete medium (DMEM+10%FBS). The cells are usually split every 2 to 3 days under sterile conditions.

2. Split the hybridoma cells to a density of 2x105 the afternoon before you plan to freeze them. They should be ~4-6x105 the next day, and have a high percentage of viable cells (>80%).

3. Prepare freezing media: 90% DMEM-10 or FBS, 10% DMSO. Cool media to 4°C, and keep on ice.

4. When cells are ready, count the cells, and pellet them (5 min ~ 1500rpm in tabletop centrifuge).

5. While cells are spinning down, label cryovials (Corning) for your cells. Indicate name of cells, concentration (if different than 10^6/mL), date, your name or initials.

6. Aspirate off old media, and replace with freezing media. The density of cells should be 1x10^6 cells/mL.

7. Aliquot cells into cryovials (~1.2 mL per tube).

8. Place vials on ice while preparing additional vials.

9. Place vials in one-half of a styrofoam rack (from a package of 15 mL conical tubes), and place the other half on top. Seal with lab tape, and put in -80 °C freezer overnight.

10. The next day, transfer your cells into a box in the liquid nitrogen tank. Complete the log located next to the LN2 tank.

(11. Save the styrofoam rack for future freezings.)