Vascular Research Division, Center for Excellence in Vascular Biology, BWH   Brigham and Women's Hospital, a teaching hospital affiliated with Harvard Medical School
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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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