Hi, I'm running an assay measuring phagocyotosis and ROS production in PBMC's. I'm using Dihydrorhodamine 123 (Thermo) to measure ROS. I then stain and fix with PFA afterwards.
I've so far read conflicting things on whether you would be able to keep your cells fixed with DHR intracellulary, or would the results be less reliable? Any insight is much appreciated!
Mari
Hey Mari,
we used Dihydrorhodamine once (not fixaded though) to measure ROS, but were not satisfied with it. First of all it diffuses everywhere (in the cell, outside the cell, over every membrane in the cell), so you can just measure "the overall" ROS in the cell. In our hands this was not useful, because in macrophages different sources are activated in different compartments.
We measured ROS with PBMCs a short while ago (but again not fixated) with our commonly used ROS probes (Isoluminol for extracellular, 6,5-Dicarboxy-DCF for cystosolic and MitoSOX for MitoMatrix ROS) with bacterial infection and this worked quite well (at least comparable with mouse macrophags).
Another concern about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. During the last years, I have measured ROS in macrophages, neutrophils and PBMCs. They react very quick (at least after infection) with ROS production and end approximately after one and a half our. This of course highly depends on the stimulus. I would always recommend ROS measurements in a plate reader with kinetics (it worked with PBMCs in our hands) instead of making "ROS-Snapshots".
We had always trouble to fix any kind of representative ROS probe for microscopy so far :(
For further information about ROS measuremtents in a plate reader please have a look at our paper (the technical conditions described there also worked for PBMCs ;)). https://stke.sciencemag.org/content/12/568/eaar5926
I hope I could help you. For any further information or question please ask.
All the best and have a nice day,
Marc
Dear Mari,
Because you are measuring a kinetic event, you need to optimize the time period you are making measurements. I suggest that you make a validation study, with 5, 10, 15, 30 and 45 minutes sample runs first. Then, optimize the time period within the results you have obtained. In our hands, with human samples optimal time is 20 minutes after we add the stimulant. The stimulant used is very important. If you are using bacteria (an ATCC bacteria line is suggested), you need to adjust your bacteria solution concentration to 1 McFarland unit. To use the same bacteria at same concentration each time is important. Also, DHR concentration is important and need to be validated. So, I suggest that you make a validation plan with consideration of all of the factors that will effect your results (fish bone analysis) and then run your samples within the plan. You will have better and more reliable results. As Marc Herb has suggested if possible to use a plate makes things easy if you have a plate reading cytometry. In my experience, to use a fixative, PFA or anything else, for kinetic assays will lead to false positive and negative results.
Best wishes,
DHR will naturally oxidise if left for any period of time. You will not be able to keep your cells for any period of time as the DHR will degrade and the results will be unreliable. The fixation process does not 'fix' the DHR. As such you cannot 'keep' your fixed cells - they need to be read immediately.
Also the question will be what are you measuring? By fixing the cells you are neither measuring the background burst rate of your PBMCs (as the fixation process will change this), nor a stimulated burst percentage.
Thank you all for your input! I have decided to leave my cells unfixed and measured the oxidative burst during my assay with a time course up to 4 hours. This found no significant changes in the MFI, so it seems to be good assay at least up until then.
Best wishes,
Mari
Hi Mari,
Relative to ROS production: working with fresh human whole blood (not PBMCs), we found considerable DHR fluorescence following PMA stimulation in monocyte and neutrophil populations by flow cytometry. Anything between 15 - 30mM DHR123 in DMSO for a final cell-stain dilution of (1:20) should be ok.
Stimulation time matters as well. A quick look at literature reveals that ROS generation is a fast process; so you may get good signal with PMA in whole blood anywhere starting from 30mins. We noticed that the fold change in neutrophil oxidative index with PMA began to drop significantly after 2h of stimulation. Hence you want to stay within that range if using PMA.
With respect to other stimulants: these affect the fluorescence intensity as well. In my hands, Mtb bacteria whole cell lysate produced a relatively higher signal than the unstimulated control under conditions stated above but significantly less than that induced with PMA.
NB: We didn't try fixing cells for later acquisition; we read immediately after the staining process.
Best wishes,
Caleb
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