Core Facilities
Cell Biology > Protocols
Freezing
Adherent Cells
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
a lid.
4. Harvest the cells
using their standard protocol (e.g. washing with Ca2+/Mg2+-free
buffer, harvesting with enzyme, and quenching with standard
medium).
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
by #5.
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
et seq.
Kay Case (10/04)
Deanna Lamont (10/04)
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