Cells dying upon temperature shift.

[Mags]

Hey guys,
The problem I have is following: I have the cells seeded in the 6 cm dish (confluency dos not seem to matter in this case). My procedure requires that I change the themperature from plus 4 C degrees (cells on ice) to pluss 37 (cells in the incubator for 15 min) and then back to 4. We use the ice cold PBS and prewarmed MEM. These cells die however, while the control cells that stay on ice only, are fine. Obviously the cells don't tolerate the temp. shift. We tried to rise and lower the temp. more gradually, but it doesn't seem to help. The cells die if I wash them with cold PBS (after incubation at 37) and if I add 37 MEM (or even 16-20 degrees PBS) to cells previously incubated on ice.
I'm wondering if anyone had the same problem. And if so...why and what can be done?

[keithbrent2000]

It's possible that the mechanical stress from pipeting in the solutions may be causing the cells to die. You may just want to put the entire dish into the incubator, and then back on ice without changing the medium.

Also, besides using control cells that stay at 4 C degrees, you may also want to have other control cells (eg pipeting in cold PBS/DMEM onto the control cells instead of the warm PBS after the initial ice incubation, putting control cells into the incubator as a second step and not performing the third step of putting it back on ice).