When I try to revive my clone from -80 degree, it looked as though there was 30-50% viability. After 3-4 hrs of revival, I found that they all are dead.
The procedure i follow:
1. Take vial from -80 degree.
2. Thaw it at 37 degree and wash the cells with complete media.
3. Centrifuge it and finally kept in new culture medium flask.
There are no contamination issues.
Hi.,
Based on my experience, if you keep your vial in -80 more than 6 months, the viability will highly reduce. In addition, I usually follow this procedure:
1. Take out the vial from - 80 C.
2. Thaw it immediately in your hands (37 C).
3. Add pre-warmed medium with serum (for the first time, usually use the high amount of serum like 10-15%).
and finally, pour it in at least two flasks.
Good Luck,
Usually, mortality is higher if DMSO is metabolized by the cells. To avoid that, you can:
1. thaw the cells very quicly by turning the tube in your hands as stated by Fatemeh Shamsabadi.
2. Don't wait for total thawing, just drop the "cell-IceCube" in a falcon tube.
containing warm medium, to dilute DMSO as fast as possible.
3. Seed the cell into a very small dish (6cm diameter) to increase cell density which increase viabilty.
3. Let the cells attach during 1-2 hour.
4. Gently aspirate medium and floatting dead cells and replace with new warm medium : it will reduced the amount of dead cells in the media which relaese toxins that can be noxious for the healthy one.
6. Replace medium every 3 days untll confluency.
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