Hey Sarah,

we measure ROS routinely in our lab (with macrophages though), but the protocol should work for all cells.

Our protocol looks at follows:

1) Centrifuge your cells (we seed them in normal 96er black culture plates)

2) Wash them once with HBSS (to remove medium)

3) Add your DCF/HBSS staining solution (10 µM works well in our hands)

4) Incubate at 37 °C for at least 15 and for maximal 30 minutes in an incubator

5) Remove the DCF solution and wash one time with HBSS

6) Add stimuli of your choice and a positive control (we use PMA, please no H2O2)

7) Fill up the wells to a total volume of 200 µl (every full clear medium is fine, no phenol red and no serum, these will quench your fluorscence)

8) Place the plate in a pre-heated plate reader and measure for at least 2 hours every minute.

And a little addition to the DCF and CellROX probes:

2',7'-Dichlordihydrofluorescein-diacetat (H2DCF-DA) is a cell-permeable derivative of fluorescein. After its diffusion into the cytosol, the two acetate groups are cleaved by cellular esterases generating 2',7'-dichlordihydrofluorescein (H2DCF). This molecule can now be oxidized by ROS to generate the highly green fluorescent 2’,7’-dichlorofluorescein (DCF). H2DCF-DA is often referred to as intracellular ROS probe. However, even after cleavage of the acetate groups, it remains diffusible and therefore does not only remain in the cytosol but also reaches cell organelles and diffuse back into the extracellular space. Therefore only total cellular ROS can be detected with this probe (Ushijima et al., 1997, Hempel et al., 1999).

CellROX oxidative stress reagents are cell-permeable fluorescent probes that are mainly used as indicators for oxidative stress. The different CellROXTM probes, however, are diffusible and cannot differ between cellular compartments and ROS subspecies, but vary greatly in usage conditions, like live cell compatibility, resistance to detergents, fixation capabilities and emitted fluorescence.

For more information, you can have a look at my "Questions section" or in our paper (which shows how ROS measuremtents with DCF look in a plate reader).

Article Mitochondrial reactive oxygen species enable proinflammatory...

I will also send a more detailed protocol and a example plate scheme over to you.

All the best and stay healthy,

Marc

Hi Sarah

We use a similar protocol as described by Marc. However, I believe the problem is really the cells that you are using. Hepatocytes for instance also have the same problem, which is displaying cellular autofluorescence. To my knowledge it doesn't exist an optimal way to solve this unfortunately. I would say try to contact the company and ask if they have something that can help. Or otherwise, if possible in your work use a more "indirect" measure like measuring GSH/GSSG levels and/or other redox parameters

Good luck!

The problem Andreia mentioned is true. Even in macrophages we observe autofluorescence, however, autofluorescence is in most cases not higher than the specific fluorescence of the probe you apply, if, and that is important, your cells produce enough ROS. And there is Andreia right again. If your splenocytes produce even with a fluorscent probe not enough ROS you won't get a signal because the autofluorscence of the cells is stronger. Like always, it depends on the cell type and the read-out. Nevertheless, give our protocol a chance and let me know how it worked.

All the best,

Marc

Maybe I can join the discussion? I'm actually hardly trying to use CellRox DeepRed as ROS-indicator in insect primary cells. And I'd like to incubate my cells before I treat them with (almost) physiological stimulus. We culture primary cells 24 h in insect medium, then wash and change medium to ringer solution with 5 µM CellRox. After about a minute we observe a signal that is continuously raising and clear distinguishable from background. I'm in doubt, if the excitation light time-lapse is to "heavy" and is the only reason, my signal is raising. And If it is ok not to wash out CellRox (signal/background ratio is ok...)

How do you excite? How often in which time?

We measure every 10 seconds (to much?) for about 5 minutes (then it is saturated). And finally compare the gradient of the control (without stimulus) vs gradient (with stimulus)

In context of this topic, I would like to bring to your attention a recent review of our group, "Functions of ROS in macrophages and antimicrobial immunity".

In this review, we give an introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense and provide an overview of commonly used ROS probes, ROS source inhibitors and ROS scavengers (also the difference between ROS scavengers and antioxidants, which are not synonymous, is explained).

If you like, please have a look at:

Functions of ROS in Macrophages and Antimicrobial Immunity

February 2021, Antioxidants 10(2):313

DOI: https://doi.org/10.3390/antiox10020313

Please also have look at our Perspective article, if you like:

Article Reactive Oxygen Species: Not Omnipresent but Important in Ma...