Hi, you probably want to first check if the cryogenic preservation and the revival are done correctly and if the revived cells are healthy, as the health state of the cell affects the adhesion: https://lab.plygenind.com/how-to-cryopreserve-mammalian-cell-lines

https://lab.plygenind.com/considerations-revive-cell-cryogenic-preservation

and then possibly adjust the media and/or surface to enhance adhesion:

https://lab.plygenind.com/factors-that-affect-cell-adhesion-in-mammalian-cell-culture

Hello Liam Cook

You may consider environmental stress as a possibility. What could be causing environmental stress? You should watch out for the following.

1. Contamination,

2. Inappropriate cell surface for attachment,

3. Temperature fluctuation in the incubator,

4. Improper gas mixture in the environment,

5. Inadequate or altered nutrients in the culture media. You may have to change the FBS which you are presently using.

After checking for each of the above, if you still fail to solve the problem, then I feel you should revisit the freezing and thawing protocols. While freezing BT474 cells, have you taken care to freeze the cells by making use of gradual freezing technique at -1°C /min? You may use Mr. Frosty freezing container. This container is designed to achieve a rate of cooling very close to -1°C/min, the optimal rate for cell preservation. Also, while thawing BT474 cells, you should apply quick thawing technique (less than 1 minute) in a 37 degree C water bath.

If you follow a good freezing and thawing protocol, you will get a good cell viability and attachment of cells to the substratum is less than 24 hours on revival. Have you checked the cell viability after thawing?

Best.