Hello,
We recently seeded a HeLa cell line purchased from ATCC, which was received over six months ago (stored at LN2). We used the following media composition: DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Sodium Pyruvate, and 1% Penicillin-Streptomycin. After approximately 24 hours, we observed that the cells had not adhered. A viability check using trypan blue indicated that the majority of the cells were dead.
In this situation, we seek advice on the best course of action to revive the cells
Hello Tanvir,
Since majority of cells have died, the probability that your culture will survive is low. Nevertheless, you may centrifuge your culture and re-seed the surviving cells in a smaller surface area culture vessel (6-well or 24-well plate) with fresh media so that the cells may come closer to each other and try to survive. Keep observing the culture everyday.
Meanwhile try to find the reason why the cells were in such bad condition.
1. Was the cell line transported in proper condition (dry ice)?
2. Did at any time during storage, the tank ran out of liquid nitrogen?
3. Did you follow a proper thawing protocol?
4. Lastly, I have observed (rarely) that one may be provided with a faulty frozen vial, in which case, you may always request the vendor for another vial.
Best.
Environmental stresses that can prevent successful attachment of adherent cell include:
- Contamination
- Incubator temperature fluctuations
- Insufficient or inappropriate cell culture surface/substrate
- Inappropriate environmental gas mixture
for more information read the article
https://www.sigmaaldrich.com/LY/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/poor-adherent-cell-attachment-troubleshooting
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