Hello
I want to investigate the effect of high temperature on ROS production in yeast. I have yeast cells which grow at 30 degree as a control and experiment cells grown at high temperature.I used Dihydrorhodamine 123 for staining and then investigated by flowcytometry and fluorescent microscope. Unfortunately, I found high signal for my control cells in flowcytometry and microscope. It’s not reasonable that control cells have more ROS at the normal temperature. I enclosed the results of flowcytometry and fluorescent microscope for my control cells here. I’ll grateful if you could give me helpful suggestions. Thanks in advance.
Hello, what was the probe dose used?
DRH123 responds to exogenous peroxynitrite and hydroxyl radical.
One possible solution is to reduce the probe dose, to get a reasonable baseline, then compare the response to a positive control.
If after getting a reasonable baseline, a positive control fails, it would suggest the probe is no longer chemically intact (has turned bad).
In addition, yeasts and certain other microbes may accumulate probe (quenched) abundantly on their surfaces, so much so that they look turned on. It is an artifact. If you introduce an ROS scavenger and the fluorescence cannot be reduced, it means it is nonspecific accumulation of lots of probes on cell surface. You may not be able to proceed further.
It is a common problem with working with small molecule probes in microbial cells.
Thanks Nai-Kei, but I didn't use any probe. I want to investigate the effect of high temperature on ROS production in yeast. I have yeast cells which grow at 30 degree as a control and experiment cells grown at high temperature. but my control cells showed high signal.
First, do it again to make sure that your results can repeatable. Second, try another probe to detect ROS, since DRH is not reliable in some conditions to detect ROS. Finally, what degree is your high temperature? Your hypothesis should be reconsidered.
pH, ionic strength, temperature, etc. could account to rising of signal. As was mentioned above DRH123 responds to exogenous peroxy radicals thus nitrite level together with low pH could account to.
Take care about the final concentration of such deluents as DMSO, EtOH, MetOH in culture medium.
You have 2 populations of cells with strong and weak DRH signal - try to test both with additional staining f.e. propidium iodide).
Finally, I'm not sure but there are different strains of yeast and optimal temperature could be also different. (I assumed you have strain but not mixture).
Thanks all you for comments.
Xing, I repeated the experiment and the results was the same. other researchers used this staining for ROS and they didn't have problem and also, now my problem is 30 degree not high temperature. I can't reduce fluorescence signal at normal temperature.
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