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I have been doing cell culture for about a year now. When I thawed my fibroblast cell line, everything went to plan. When I checked them a day later, they were all dead (<3-5% confluent). I'm not sure what happened since the media is fine, the incubator is fine and I didn't change anything in the protocol. I have used this protocol dozens of times before. Any tips or ideas of what could have happened? The only things I can think of are a mistake that I didn't realize or an error in the freezing process...

EDIT: My thaw protocol is the following: add warmed growth media and incubate until cells are thawed. Then transfer into 15mL conical tube followed by 3mL warmed media (used to wash out cryo vial). Centrifuge to pellet cells. Remove supernatant and resuspend pellet in fresh (warm) media. Place in a T25 flask and incubate.

This is all that I have done to the cells.

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    $\begingroup$ If you post you thaw protocol and how you treat the cells further, we can possibly help you. $\endgroup$
    – Chris
    Commented May 20 at 17:00

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