Cells dying upon temperature shift. - (Jan/27/2008 )
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?
What kind of cells do you use?
Well...These are PAE (Porkine Aortic Endothelial) cells, stably transfected wiht one receptor. But it seems like other clones of PAE cells are less sensitive to the temperature shift.
So, do you wash the control cells as well? Do you have a treatment with a chemical at 37? Dying means detaching from the plate?
wat r the incubation time in 37/4/37C?
in general after 4C treatment for couple of minutes does not harm cell too much, when u put them in warm medium, they adapt quite easily.
as mentioned by zek, if u observe this phenomenon only in transfected cells but not in control cells, then some connection exist between ur transfected protein and temprature sensitivity of PAE.
To Zey: Thanks, girl Hope you're not working too much!
I don't treat with any chemical. I just put the cells at 37 to induce endocytosis...and yest I do wash the control cells, but only with the 4C solutions. And what I mean by dying...tehy do not detach...not most of them, but they shrink, round up and completely lose they original shape...Impossible to look at them in the microscope.
To Dr.House: Thanks! The cells taken out from the 37 are put on ice, washed with ice cold PBS (as long as I let them first cool down on ice slowly, before I treat them with cold pbs they seem ok). Then I incubate them 30-40 min at 4C. then I'm supposed to have teh cells at 37 for 15 min...and that's when most of them shrink (round up completely) and some of tehm detach. After 37C they are back at 4C until I fix them. Oh...and both the control and treated cells are transfected.
once i was working with HAEC, in PBS solution they behave quite strangely, \
i saw the shrinkage when cells are in 6 well plate/on ice/ PBS for 15-20 minutes.
after that i did not use the PBS rather simple EBM without growth factors, afterwards they looked completely ok for long time.
i suggest u to give a try
let us see wat happens.
Thanks a lot! I'll give it a try