Cell Biology > Protocols
1. Make a freezing medium
by supplementing the cells' regular growth medium with
10% DMSO and increase the amount of serum in the medium
to 20%, at least.
2. Chill the freezing
medium to ~ 0 - 1°C in an ice water bath.
3. Prepare an ice bucket
with crushed dry ice (~ 1-2 inch layer) and cover with
4. Harvest the cells
using their standard protocol (e.g. washing with Ca2+/Mg2+-free
buffer, harvesting with enzyme, and quenching with standard
5. Count the suspended
cells, if desired. Cells freeze most efficiently at
concentrations of between 1 and 10 million cells /ml
(final suspension in freezing medium). Alternatively
each 75 cm2 flask can be divided into 2-3 vials containing
1 ml each of cells suspended in freezing medium.
6. Centrifuge the cells
at 200G to pellet them (~5 minutes), at 4°C if possible.
7. Discard the supernatant
and resuspend the cells in freezing medium as determined
8. Aliquot the new cell
suspension into cryovials, and label the vials as required.
9. Put the vials into
an ice-water bath to chill the cells to 0-1°C or
less (~15-20 minutes).
10. Tighten the tops
and transfer the vials to dry ice, for snap-freezing.
11. When frozen solid
(3-4hrs), transfer the vials to liquid nitrogen storage
tightening tops one more time.
12. For non-adherent
cells, make a freezing medium, as in #1 (serum concentration
can be increased if needed) then proceed to step #5
Kay Case (10/04)
Deanna Lamont (10/04)