I have heard it argued that you should get the maximum pixel intensity (but won't that be prone to artifacts?), because that is the maximim ROS burst. I have also heard you should rather count the number of pixels that are above a threshold intensity, which is maybe a measure of number of bursts. There is also the simplistic total intensity of the image (or total intensity of the region of interest of a fixed sized area) or average intensity in the region of interest (so that you can have different sized areas). And I also heard an argument for getting the average intensity of all of the pixels that fall above a certain threshold. So what is the most widely trusted method for mitosox? In my case, if there is a negative result (no change in mitosox for my mutant of interest) then this will be very informative - but I don't want to be mislead by the quantification having produced an artificial negative result because it wasn't calculated appropriately.
Live cell imaging of Mitosox staining is very difficult to quantify. Getting reproducible results is almost impossible. The best method to quantify Mitosox staining is by flow cytometry. We have used this in our laboratory to see differences between WT and respiration deficient cell lines.
I typically co-label nucli with hoechst and my mitochondria with a probe like MitoSox; in cells grown in 96-well format.
I then visualize center field at x10 or x20 for both dyes. After background subtraction I count the number of nucli in the field and total ROS signal per field. Then I compare the ROS/nucli levels to an n>5.
You can also find the average ROS/cell in your control and count the number of cells which have something like 4x the ROS/cell level of labeling and state that this is what you concider 'activated', then count total and activated number and express a percentage.
The key is to have big n numbers, which is why 96-well format is so great.
Live cell imaging of Mitosox staining is very difficult to quantify. Getting reproducible results is almost impossible. The best method to quantify Mitosox staining is by flow cytometry. We have used this in our laboratory to see differences between WT and respiration deficient cell lines.
Thank you all for your advice. It seems like nobody in this group is recommending the method of getting the maximum intensity, nor the method of counting pixels above a threshold, but rather simply looking at averages.
Martyn, thank you for your suggestion of counting number of cells that are above a threshold - that was not in my original list of possible quantification strategies, and I will see how that result compares.
Mercy, I appreciate your suggestion of flow cytometry, but for several reasons, in my experiment, this is not possible.
Hello Beverly.
I've been using MitoSOX a long time ago, but I always performed the analisys of cell populations by flow cytometry. What kind of cells do you work with? Is it possible for you to harvest the cells and analize them by flow cytometry? If it is so, you can easily identify cells bearing superoxide-producing mitrochondria. Evenmore, you can quantify the levels of production by the MFI of possitive cells.
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