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Heat-shock treatment problems with adherent cells - (Oct/29/2008 )

Hi all, I am having issues with doing a heat-shock treatment (42-45˚C, 1 hour) on HEK293 cells. When I trypsinize and put into conical tubes for heat-shock treatment, even 0 hours (which is just trypsinized + 1 hour for drug treatment), HSP90 is shown to be induced, with no change for each time point. Supposedly, HSP90 is supposed to be induced upon heat-shock treatment, so the constant levels baffled me. So I wondered if the trypsin was introducing mechanical stress that would cause HSP90 to be induced prematurely (note: I did this twice). Thus, I kept the cells adherent on 5-cm plates, wrapped them in Parafilm, and submerged them in a waterbath, using glass flasks to keep them submerged. One hour after the heat-shock treatment was finished and I put them in the 37˚C incubator, I noticed that the cells in all the plates were floating, indicating that they were dead. Can anyone tell me what is the right way to do a heat-shock treatment on adherent cells?



You want to do heat shock treatment on adherent cells at 42 to 45degreeC for 1 hr. You have a control w/o treatment at zero hour.
Are you looking at protein or mRNA level (just curious)?

For the control: I wouldn't trypsinize the cells at zero hour, I would just isolate either the protein (lysate) or mRNA right away. Then keep at appropriate storage temperature.

For the treatment cells: I would prefer to place adherent cells (in culture flask) inside an incubator (without CO2 is fine - just less buffering that's all) with temperature set to 42 to 45degree C and start timing until an hour. Then proceed to isolate protein (or mRNA) right away, without trypsinization.

NOTE: (1) The isolation step, I would spin-wash the cell with PBS prior to lysis step.
(2) Floating cells might not really indicate dead cells (probably just shocked, though some might die due to the stress), but if you really want to be sure, aliquot some for trypan blue exclusion assay.
(3) If you find that cells are all dead due to treatment, suggest to your PI to include an acclimatization step to the protocol, i.e. slowly, or in phases, increase from 37degree optimal to 42degree. Though you want to shock the cell, but not to the extent of killing it ya? So acclimatization step sometimes is crucial.

Best of luck.

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