FREEZING OF CELLS
1. Make “Freezing Medium” (70% of the usual growth medium for cells; 20%
FBS; 10% DMSO; no antibiotics)
2. Dissociate Cells, Spin them down, and reconstitute in Growth Medium.
3. Count cell density on hemocytometer.
4. Calculate the volume of Freezing Medium to add to the cells to get between 1
and 5 million cells /ml (depends on the cell line)
5. Spin down the cells once again.
6. Add appropriate amount of Freezing Medium to resuspend the cells.
7. Aliquot the cells into vials ( between 1 and 1.5 ml per vial depending on the vial)
8. Put the vials on ice and leave there for 15 minutes.
9. After 15 minutes, place the vials in a Styrofoam container and place in a -20°C
freezer for 2-3 hours.
10. Move vials in Styrofoam container to a -80°C freezer and leave overnight.
11. After 24 hours, move the vials into the liquid nitrogen cryotank (keep vials in
Styfoam when moving so they don’t thaw).
12. Can check viability by thawing one tube.
THAWING OF CELLS
1. Remove cells from Liquid Nitrogen Tank and indicate on log which vials were
thawed.
2. Keep the cells frozen until the next step.
3. Thaw the cells in the vial rapidly but only partially (ice pellet separated from side
of tube) by holding in hand or in placing briefly into a 37°C water (or dry) bath.
4. Immediately add contents of the tube (by dumping it) into a 50 mL tube
containing ~40 ml of prewarmed Growth Medium, cap tube and mix by inverting
a couple of times.
5. Centrifuge the cells at 1000 rpm for 5 minutes.
6. Remove the supernatant.
7. Add the desired volume of Growth Medium (usually 20 mL DMEM with 10%
FBS plus pen/strep) to the pellet of cells and mix by carefully inverting.
8. Transfer the reconstituted cells to a flask, and place the flask in an incubator.