I apologize, it was ThermoFisher, not ATCC. If you look at page 2 of the manual it says they do not recommend warming the reagents in the subculture section. Perhaps it's just trypsin? But they say reagents with an S. And I've tried the room temperature trypsin protocol and it doesn't work even after 10 minutes.

https://www.thermofisher.com/order/catalog/product/C0055C

Footnote sez "Damage to cultured HEKa can occur during trypsinization. This damage may result from exposure of the cells to the Trypsin/EDTA solution for excessive lengths of time, trypsinization at temperatures exceeding room temperature and/or excessive mechanical agitation. Check to make sure that the temperature of trypsinization is appropriate and, if necessary, alter the incubation time of the procedure." I've used several oral keratinocyte cell lines and have not seen this warning before. I incubate ~10 min in CMF-PBS at 37 C followed by 0.05% trypsin/EDTA at 37 C for ~5 min.

@Thomas, thank you, I didn't see that footnote. I use a very dilute concentration of trypsin and also incubate at 37C. I've tried at room temperature but they just don't detach. I could use a much higher concentration of trypsin at RT, but then doesn't that defeat the purpose of not exposing them to a ton of trypsin? I'm not sure what it is about the trypsin that would damage the cells. I haven't noticed them being damaged.

@Andrea, thank you for your insight. My cells are primary/secondary cultures (non-transformed) but also stick very well to plastic and glass. I have indeed tried to incubate with trypsin at RT and even after 10 minutes almost 100% are still attached. I can wash almost an infinite number of times with PBS after my treatments and they are still stuck. I don't think I've observed damage after the 37C trypsin Tx, but I don't know what that "damage" would even look like. I always neutralize the trypsin and then count the cells using trypan blue and always see over 90% viability and I think they look okay under the microscope. I appreciate all the other suggestions for detachment in case I need to use them in the future!

I have always asked myself: Do I really need to pre-warm my culture media while sub-culturing the cells? What would be the impact of the few minutes of lower temperatures towards my cell culture considering the big picture, since soon it will go back to 37 C during incubation? For trypsinization, in my case (for A549 cells) I do need to keep the cells with trypsin for 5 to 7 min at 37C to get them in suspension. However, when it comes to change culture medium only, my cells grow fine when I use non pre-warmed media. In my view, avoiding the pre-warm is beneficial because you eliminate 1 variable of contamination. If you use a water bath and don't strictly monitore the water conditions, depending on how well you wipe the flasks, you can bring some nasty stuff inside you hood!