We are performing plate based methods for detection of ROS in LPS-induced RAW264.7 macrophages. We used dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that after diffusion in to the cell is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescin (DCF). DCF is a highly fluorescent compound that is detected by fluorescence (excitation:emission::495nm:529nm). Black well plates are generally preferred for assay to reduce interference during reading.

We commonly use DCF on stimulated endothelial cells (HUVEC), dissociate them and analyze by FACS. Can send you a protocol if helpful. It's easy and inexpensive :)

We are performing plate based methods for detection of ROS in LPS-induced RAW264.7 macrophages. We used dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that after diffusion in to the cell is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescin (DCF). DCF is a highly fluorescent compound that is detected by fluorescence (excitation:emission::495nm:529nm). Black well plates are generally preferred for assay to reduce interference during reading.

Mostly we use DCF for peroxynitrite or H2O2 measurement and DHE/mitoSox for superoxide measurement. I have read using them in FACS. You may give it a try.

I'll echo Ru-Jeng Teng's comment and add that it can be done by Flow Cytometry. You can find the spectra for each to ensure you can use them on your instrument at the Life Technologies web site.

We measure intracellular ROS by carboxy-H2DCFDA and mitochondrial superoxide anion by MitoSOX (Molecular probes) in different cell cultures. Both methods are reliable and I think they can work well also in macrophages.

If for me, i prefer dihydroethidine (DHE) dye and detect by flow cytometry. DHE measure superoxide anion level, and relatively it is more stable and reproducible as compare to DCF. DCF may diffuse to extracellular environment and cause false-negative result.

Flow cytometry for sure is more powerful than plate reader. It measure until single cellular stage instead of whole cell population. By using flow, you can get back the plate -reader result by refer to the mean fluorescence value. You can quantify the percentage of cells population which showed high level of ROS.

I was using DCF for my work and diferent dye specific to the ROS like SOSG for singlet oxygen.

https://www.researchgate.net/publication/225081695_Rakesh_Kumar_Sinha_Josef_Komenda_Jana_Knoppov_Michaela_Sedlov__Pavel_Pospil_Small_CAB-like_proteins_prevent_formation_of_singlet_oxygen_in_the_damaged_Photosystem_II_complex_of_the_cyanobacterium_Synechocystis_sp._Plant_Cell_and_Environment_%282012%29_35_806818

Article Rakesh Kumar Sinha, Josef Komenda, Jana Knoppová, Michaela S...

there are some ready kits available for detecting ROS by flow cytometry and ELIZA. I prefer flowcytometry especially when combined with viability dyes to discriminate the viable from apoptotic cells. This will provide more powerful and valuable information. You may be able to find commerical kit however you have to define your probes and procedure to serve your lab goals and capacity. Please find Mahfouz et al 2010 (https://www.researchgate.net/publication/23178052_Evaluation_of_chemiluminescence_and_flow_cytometry_as_tools_in_assessing_production_of_hydrogen_peroxide_and_superoxide_anion_in_human_spermatozoa) Thanks.

Article Evaluation of chemiluminescence and flow cytometry as tools ...

Do not use DCF, is "dirty" probe for ROS detection is better to HE based probes.Cells are incubated with DHE or mitoSOX (5 μM) for 30 min and their oxidation is monitored by flow cytometry using FL-2 (488 nm ex, 585/42 nm em) ROS, because dihydroethdium can be oxidized by horseradish peroxidase (HRP) and H2O2 with the formation of E+ and this apply for MitoSOX.

Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vásquez-Vivar J,

Kalyanaraman B. Superoxide reacts with hydroethidine but forms a fluorescent

product that is distinctly different from ethidium: potential implications in

intracellular fluorescence detection of superoxide. Free Radic Biol Med. 2003 Jun

1;34(11):1359-68.

There is some controversy surrounding the use of DHE, as there is a concensus that the prode must intercalate with DNA to fluoresce in response to ROS properly - I'm not sure where I stand with this probe.

May I ask - are you looking at ROS specifically within the phagosome / generated extracellularly / within the cytoplasm? Much of my PhD was concerned with the development of diagnostic tools for ROS monitoring, I used NR8383 macrophages as my major model. You might want to consider DCF-DA or DHR-123 perhaps with dextran conjugation for phagosomal monitoring however be aware of the pitfalls of these probes (Boulton et al, 2011 DOI: 10.3109/10715762.2010.517751 - sorry for the self citation).

You might also be interested in taking advantage of ROS-sensitive nanosensors that can be used to monitor ROS in a quasi-quantitative manner (Henderson et al, 2009 DOI: 10.1016/j.bios.2009.05.029) these are pretty easy to fabricate. If you’d like to know more, feel free to PM me.

Also - I'm a fan of my plate reader over the FACS analysis for the kind of work I do. The plate reader is cheaper, faster, easier to prep for and monitors adherent cells in a more 'native' state (them being attached to the bottom of a plate and all!) However for you (and indeed in the past for me), the macrophages are essentially non-adherant so this isn't so much of an issue. I feel that FACS has it's place in generating a high volume of data from a relatively low population of cells, and being able to gate out dead cells is a real bonus, however the process is incredibly time and money consuming and for my work it simply cannot be justified. I tend to reserve the use of FACS analysis for projects where only FACS will do, for example, where I'm unsure of the impact of cell death upon the data I'm generating in UV studies, however this is a rarity within my work so my platereader generally does just fine.

@SaraJayne: Thanks for the objective consideration of the dyes and methods. As I was a bit naive about measuring ROS, I was considering measuring it in an as general way as possible. However, the possibility to localize the dye to phagosomes opens up a much more relevant approach to my question. Will have to read up on those pitfalls, thought. I guess in the end I´ll go for measuring "general" (cytoplasmic) and phagosomal ROS.

Interesting to read that your macrophage line was not adherent. Bone marrow derived macrophages as well as monocyte derived macrophages are extremely adherent and it can be quite difficult to detach them - there is even a thread on this here at RG :-) So probably I´ll go for a plate-reader based solution.

@all: thanks for the many different answers. Now I´ll have to do some more pondering...

@Christoph You're more than welcome. My macrophages are rat alevolar macrophages (non-primary) and these are the only ones I've had the pleasure of working with - certainly not my area of specialty! I'm a greater fan of adherent cells for what it's worth.

If you need any advice in the future regarding ROS monitoring do feel free to get in touch. Good luck!

Our current experiments involve mouse BMDMs which were genetically modified to be YFP and EGFP. This, as well as autofluoresence issues in macs precluded the use of many of the common "green" reporters for ROS. Fortunately, we have a LSRII Fortessa so we were able to put our live-cell ROS-reporter in on Deep red (em 665) around the APC channel. We used the Molecular Probes/Invitrogen CellROX Deep Red http://products.invitrogen.com/ivgn/product/C10422 according to the batch sheet. It worked nicely and we were able to detect a half-log shift in living (Invitrogen, Violet Live/Dead) total-cell ROS production by a 100ng/ml ultra-pure LPS stimulation for 8h (Red laser @ 633nm, 40mW). We feel the LPS induces only a "modest" mitochondrial-sourced ROS burst a la West et al., 2011 Nature. Guessing, if respiration inhibitors or things like Rotenone were used there could be greater ROS production and the MFI shift would be greater. We also compared the ability to re-use an opened tube once after opening it only briefly. It did not work and unless your ROS is massive (read neutrophils) we feel it can only be used once.

Best of luck !!

@ Steven:: I found it hard to monitor intracellular ROS generation in real-time stimulated by LPS too, however when we wanted too look at the capacity for extracellular generation through NADPH oxidase as a function of phagocytosis, LPS was a great stimulant.

As far as I am aware mitochondrial ROS as a consequence of LPS stimulation is not a proximal effect, rather is is a secondary response to the initial insult. For mitochondrial ROS generation, antimycin A gave the largest ROS response for us(Henderson et al, 2009 Free Rad Res) due to the bilateral generation of superoxide (St-Pierre et al, 2002 J Biol Chem) upon inhibition of Complex III (bear in mind this work was done in isolated mitos with a superoxide selective electrode, but it did give a really clear picture in terms of superoxide generating capacity) . By inhibiting with rotenone at complex I we observed a smaller flux of superoxide in comparison as electrons were leaked only to the inner matrix as a consequence, an incredibly difficult place to monitor ROS in real time in live cells!

Doesn't stop us trying though, does it? :)

@ SarahJayne: We were using our CellROX as a "global red detector" of ROS (probably many types) in our living cells. We have good flow equipment and I feel comfortable with those types of assays. It took some kinetic work but we got it to work 4 times out of 5 experiments (Triplicate samples each exp). In work yet to be published from our lab we did not see any ROS production from activating a different innate immune receptor family member but our LPS positive control worked just fine, although not dramatic. 7.5hr stimulation, add the CellROX, wait 30 min, wash 3x and assay on livings. // I agree with your extracellular comment. Assays also capturing NADPH-sourced extracellular ROS things could be much easier to detect. For the questions we were asking from our macs we were interested in ROS inside the cell though. Hopefully in a few months we can get our work in press and you'll understand our angle a bit better.

Cheers !!!

FYI: Example Living cell ROS Histogram. Grey shade Unstimulated, Solid line Stimulation X, Dotted line Stimulation 8hr Ultra-pure LPS (Invivogen) 100ng/ml.

>40,000 living cells gated, singlets.

@SarahJayne and Steven: Great discussion here :-) I am very grateful for this, it´ll reduce the time I´ll need to fiddle around and establish this technique in our lab.

In our case, we´re going to compare BMDMs form WT and KO-mice, the most physiological condition will actually be to measure ROS inside phagosomes. As far as we can tell there is no difference in phagocytosis (at least of beads) -a prerequisite for such measurements. Therefore I´ll go for dyes coupled to the same type of beads I used for phagocytosis.

Found this review: maybe also interesting... http://www.tswj.com/2011/741046/

Oh Hey! Nice work! Looking forward to seeing the article! I've not used CellROX before - definitely one for me to check out. Thanks for the heads up!

Hi everyone..I am quite new to this ROS measurement assays. I am here with few of my queries. Hope I will find some suggestions;

What is the right way of using H2DCFDA probe (Pre or post addition of the probe??) for the detection ROS in macrophages if the effects of drugs on the ROS production have to be evaluated in vitro by flow cytometry?. Since ROS molecules are short lived, post addition of this dye will be effective enough? I am adding the probe prior to any treatment to my cells, but since minimum washing is recommended, i am wondering if this may have altered the outcome. I do not see much of a difference in ROS between groups treated or not with the drugs. I also see strong autofluorescence which makes it quite hard for the analysis. Valuable suggestions are welcome. Thank you

Hi everybody. Great discussion. So far, we have good results with mitocondrial superoxide using MitoSox Red with FACS Canto in HEK cells, as well as CM-H2DCFDA for whole cell ROS measurements. But here comes the question: does anyone know a dye that does not cover the red and/or green laser and filter (PE, APC and FITC)?

I have used this kit recently, ENZO total ROS detection kit (ENZ-51011). I will recommend to go for this, but you can first evaluate under confocal microscopy before FACs. That will give you idea for flow cytometer, because you need enough intensity and be insure that it is perfectly working. you can also ask me if you need help in your methodology.....

......

Hi all. I am just reading this discussion over the measurements of ROS on macrophages and have a couple of questions as I am trying to interpret my data and why the assay I have used did not work out and I would be very happy if some of you could get back to me.

@Steven: I have used to detect ROS production in naive monocytes as well as macrophages the CellROX Deep Red from Molecular probes that apparently it is possible to be used for fluorescent detection as well as for imaging analysis and FACS as well.

In the lab, I've tried in 3D LPS stimulation 100 ng/mL for 30 min and did not get any good results.

Then we tried to use in 2D 200 ng/mL for 24h stimulation.

I saw you mentioned you have used 100ng/mL for 8h.

Do you have any idea about this? Did you ever tried to use this CellRox and measure fluorescence only? Thanks!  

Hello

Does anybody ever used the cellrox (green) with the mitosox in parallel by facs?

Thanks

Hello,

I would like to mesure superoxyde production in neutrophils using DHE probe by flow cytometry. Which channel do i have to use ? I think emission of DHE is about 620 nm..?

Thank you for your answer