The authors would like to thank other members of the Tigno-Aranjuez Lab including Madelyn H. Miller, Omar Cardona, Andjie Jeudy, and Roopin Singh for their help in laboratory upkeep and mouse colony maintenance. Support for this research was provided by grant R00 HL122365 and Start-up funds to J.T.T-A.

<ol><li>Winterbourn, C. C., Kettle, A. J., Hampton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+Oxygen+Species+and+Neutrophil+Function%5BTitle%5D)+AND+%22Annual+Review+of+Biochemistry%22%5BJournal%5D)">Reactive Oxygen Species and Neutrophil Function.</a> Annual Review of Biochemistry. 85, 765-792 (2016).</li>
<li>Schieber, M., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROS+function+in+redox+signaling+and+oxidative+stress%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">ROS function in redox signaling and oxidative stress.</a> Current Biology. 24 (10), R453-R462 (2014).</li>
<li>Robinson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+oxygen+species+in+phagocytic+leukocytes%5BTitle%5D)+AND+%22Histochemistry+and+Cell+Biology%22%5BJournal%5D)">Reactive oxygen species in phagocytic leukocytes.</a> Histochemistry and Cell Biology. 130 (2), 281-297 (2008).</li>
<li>Thomas, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+phagocyte+respiratory+burst%3A+Historical+perspectives+and+recent+advances%5BTitle%5D)+AND+%22Immunology+Letters%22%5BJournal%5D)">The phagocyte respiratory burst: Historical perspectives and recent advances.</a> Immunology Letters. 192, 88-96 (2017).</li>
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<li>Iles, K. E., Forman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+signaling+and+respiratory+burst%5BTitle%5D)+AND+%22Immunologic+Research%22%5BJournal%5D)">Macrophage signaling and respiratory burst.</a> Immunologic Research. 26 (1-3), 95-105 (2002).</li>
<li>Curnutte, J. T., Whitten, D. M., Babior, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+superoxide+production+by+granulocytes+from+patients+with+chronic+granulomatous+disease%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Defective superoxide production by granulocytes from patients with chronic granulomatous disease.</a> New England Journal of Medicine. 290 (11), 593-597 (1974).</li>
<li>Good, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatal+%28chronic%29+granulomatous+disease+of+childhood%3A+a+hereditary+defect+of+leukocyte+function%5BTitle%5D)+AND+%22Seminars+in+Hematology%22%5BJournal%5D)">Fatal (chronic) granulomatous disease of childhood: a hereditary defect of leukocyte function.</a> Seminars in Hematology. 5 (3), 215-254 (1968).</li>
<li>Holmes, B., Page, A. R., Good, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+of+the+metabolic+activity+of+leukocytes+from+patients+with+a+genetic+abnormality+of+phagocytic+function%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function.</a> Journal of Clinical Investigation. 46 (9), 1422-1432 (1967).</li>
<li>Windhorst, D. B., Page, A. R., Holmes, B., Quie, P. G., Good, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pattern+of+genetic+transmission+of+the+leukocyte+defect+in+fatal+granulomatous+disease+of+childhood%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">The pattern of genetic transmission of the leukocyte defect in fatal granulomatous disease of childhood.</a> Journal of Clinical Investigation. 47 (5), 1026-1034 (1968).</li>
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<li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Introduction+to+Reactive+Oxygen+Species%3A+Measurement+of+ROS+in+cells%5BTitle%5D)+AND+%22White+Paper%22%5BJournal%5D)">An Introduction to Reactive Oxygen Species: Measurement of ROS in cells.</a> White Paper. , (2015).</li>
<li>Woolley, J. F., Stanicka, J., Cotter, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+reactive+oxygen+species+measurement+in+biological+systems%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Recent advances in reactive oxygen species measurement in biological systems.</a> Trends in Biochemical Sciences. 38 (11), 556-565 (2013).</li>
<li>Chen, X., Zhong, Z., Xu, Z., Chen, L., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((2%27%2C7%27-Dichlorodihydrofluorescein+as+a+fluorescent+probe+for+reactive+oxygen+species+measurement%3A+Forty+years+of+application+and+controversy%5BTitle%5D)+AND+%22Free+Radical+Research%22%5BJournal%5D)">2',7'-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy.</a> Free Radical Research. 44 (6), 587-604 (2010).</li>
<li>Zielonka, J., Kalyanaraman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydroethidine-+and+MitoSOX-derived+red+fluorescence+is+not+a+reliable+indicator+of+intracellular+superoxide+formation%3A+another+inconvenient+truth%5BTitle%5D)+AND+%22Free+Radical+Biology+and+Medicine%22%5BJournal%5D)">Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth.</a> Free Radical Biology and Medicine. 48 (8), 983-1001 (2010).</li>
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The authors have no conflicts of interest to disclose.

DCFH2-DA and DHE-based detection of ROS is a widely-used technique14,15. Ease of use and the adaptability of these ROS probes for kinetic microplate formats, fluorescence microscopy or flow cytometric analysis has contributed to their popularity. However, in our studies of Fc&#947;R-mediated macrophage functions, there did not seem to be a standard protocol for performing this assay for flow cytometric analysis of Fc&#947;R cross-linked cells. Given the reactive nature of the analytes being assessed, we have found that timing is of crucial importance in making sure that reproducibility is achieved. Although we have also performed kinetic microplate assays, there can be a wide variability in the intensity of fluorescence from experiment to experiment, making it difficult to aggregate biological replicates for statistical analysis. Flow cytometric use of these probes allows for quantification of &#8220;ROS positive&#8221; cells which resolves some of these issues but is not without complications. This is especially true when using a flow cytometer equipped with an autosampler. In this case, analysis of a large number of samples will impact the amount of time the cells are exposed to various stimuli. To mitigate this, we have incorporated the lag time in between sample analysis into the protocol to make sure that the analysis is performed on cells stimulated for a similar amount of time.
Another important factor in flow cytometric ROS analysis is the inclusion of appropriate controls. It cannot be overstated that every experiment must have (at least) all the experimental and compensation controls we list in this protocol. These are important to ensure the validity of each experiment as well as to be able to justifiably exclude samples if needed. Some examples of this include if macrophages did not differentiate/mature appropriately and exhibited much lower ROS production even with inducer treatment. Another case could be inappropriate activation of macrophages even prior to Fc&#947;R cross-linking, resulting in a high basal ROS signal. Use of ROS inhibitors in conjunction with specific Fc&#947;R cross-linking showing disappearance of fluorescent signals is another important control we have often found lacking in other studies performing similar assays. The ROS inhibitor used in this study, N-acetyl-L-cysteine, is regarded as a universal ROS inhibitor. However, if one is interested in ROS produced by specific enzymes, other specific ROS inhibitors can be included in the assay as well. Some examples include mefanamic acid (cyclooxygenase-dependent ROS inhibitor), apocynin (NADPH oxidase-dependent ROS inhibitor), or allopurinol (a xanthine oxidase-dependent ROS inhibitor).
It is additionally crucial to understand what is actually being measured in this readout. As previously mentioned, DCFH2-DA measures multiple ROS species, and so, fluorescence resulting from the green probe cannot be used to discriminate one ROS species from another14. Likewise, the orange probe (likely DHE), although often cited as superoxide specific, can produce both superoxide-specific and superoxide-independent oxidation products, which cannot be distinguished without the use of additional techniques such as HPLC15. However, for the purposes of measuring &#8220;total ROS&#8221; or &#8220;induced ROS&#8221; during the respiratory burst, use of these probes alongside the appropriate controls, may be acceptable. Many new fluorescent based ROS sensors have emerged or are being developed11,13. Some, such as the redox-sensitive fluorescent proteins, have the advantage of a dynamic measurement of ROS production due to their reversible oxidation. However, these would require genetic manipulation of cells, which may not always be feasible or desired. In many cases, fluorescent probe-based ROS detection is still a valid and useful tool, as long as the experiment is standardized, well-controlled, and the conclusions derived from such experiments are not overstated.
The benefits to using this commercially available ROS kit is that all necessary reagents including ROS inducers, scavengers, and titrated probes are included and &#8220;ready-to-use&#8221;. If the period of experimentation is anticipated to be brief (completed within a week), this kit can provide a more cost-effective method for performing flow cytometry-based ROS detection without the need to buy or optimize each specific component individually. We additionally demonstrate using this kit with a specific agonist (Fc&#947;R stimulation) and demonstrate reproducibility across experiments; we assess effects of priming times on ROS generation; we compare using similar probes, inhibitors, and inducers from different companies; we provide examples of unsuccessful experimentation; and lastly, we combine cell-surface staining with use of these ROS probes. This would potentially allow for simultaneous detection of ROS from different cell types within a mixed population, requiring less sample and reagents. For example, this might be particularly useful when differentiating between macrophage and neutrophil-derived ROS, the main cell types capable of responding to Fc&#947;R ligation. For multiple experiments which have to be performed with long intervening periods in between, use of a kit might not be as ideal. Multiple aliquots of the probes are not provided and once reconstituted, the manufacturer only recommends that reagents be used within a week (as ROS probes are highly reactive). If it is anticipated that this period is incompatible with experimentation, we recommend that individual reagents rather than a kit be purchased separately and reconstituted only during the day of experimentation. As we demonstrate here, further optimization of the individually purchased components may additionally be needed prior to the assay. Overall, we hope that this work provides a useful resource for researchers using flow cytometry to reproducibly measure ROS generation.

Reactive oxygen species (ROS) are reactive molecules or free radicals that are by-products of aerobic respiration (reviewed in 1). These include the superoxide anion, peroxide, hydrogen peroxide, hydroxyl radical, and hydroxyl ions, among others. Under normal physiologic conditions, ROS are produced mainly by the mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and are rapidly detoxified by various enzymes and proteins such as superoxide dismutase and glutathione. An exaggerated production of ROS or a defect in the ability to remove ROS can result in oxidative stress, whereby reactive oxygen species promote the damage of proteins, lipids, and DNA leading to cellular stress or death and pathological disease states. However, it is currently appreciated that ROS can also act as signaling molecules (redox signaling), and ROS-mediated modification of various molecules and pathway intermediates can influence cellular metabolism, proliferation, survival, inflammatory signaling, and aging2. In phagocytic cells, ROS plays an essential role in providing antimicrobial activity during the so-called &#8220;respiratory burst&#8221;1,3,4,5,6. During the response of phagocytes to external stimuli, components of the NADPH oxidase complex (p40phox, p47phox, p67phox) translocate from the cytosol to the phagosomal membrane containing the gp91phox and p22phox subunits, and together with the actions of Rac1/2, form a fully functional NADPH oxidase enzyme complex. The assembled NADPH oxidase then utilizes NADPH to reduce oxygen to superoxide within the phagosomal vacuole. Superoxide anions can directly cause damage or be dismutated into hydrogen peroxide. Both superoxide and hydrogen peroxide can react with other molecules to generate highly reactive hydroxyl radicals. Damage is mediated by reaction of these ROS with iron-sulfur clusters on proteins or by causing base oxidation of DNA, ultimately leading to restricted microbial metabolism or death of the microbe5. The importance of the NADPH oxidase enzyme complex and ROS produced during the respiratory burst is illustrated clinically in patients with Chronic Granulomatous Disease (CGD)7,8,9,10. Individuals with CGD possess mutations in gp91phox, resulting in a lack of ROS production and susceptibility to recurrent infections with bacteria and fungi which are not usually a concern with immunocompetent individuals. Therefore, whether studying oxidative stress, redox signaling, or host defense, being able to measure ROS production in real-time is a useful endeavor.
Multiple assays have been utilized to measure ROS production or the results of oxidative stress11,12,13. Among these, one of the most widely used is the fluorescent probe 2&#8217;,7&#8217; dichlorodihydrofluorescein diacetate (DCFH2-DA)14. This molecule is colorless and lipophilic. Diffusion of DCFH2-DA across the cell membrane allows it to be acted upon by intracellular esterases, which deacetylates it into DCFH2, rendering it cell impermeable. The actions of multiple types of ROS (hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radicals) on DCFH2 oxidize it into DCF which is fluorescent (reported Ex/Em: 485-500 nm/515-530 nm) and can be detected using a flow cytometer equipped with a standard filter set for fluorescein (FL1 channel). Superoxide does not strongly react with DCFH2 but can react with another probe dihydroethidium (DHE) to yield the fluorescent product 2-hydroxyethidium (as well as other fluorescent superoxide-independent oxidation products)15. The fluorescent products of DHE oxidation can be detected using an excitation wavelength of 518 nm and an emission wavelength of 605 nm (FL2 channel). Although relatively simple to use, utilization of these probes for detection of ROS requires knowledge of their limitations and careful incorporation of staining procedures and controls into the specific assay being performed in order to have valid experimental results and conclusions. The following protocol demonstrates the use of a commercially available kit employing these 2 probes designed to measure ROS by flow cytometry. We stain primed bone marrow-derived macrophages with these probes and induce ROS production through Fc&#947;R cross-linking. We present representative data obtained using this protocol and stress appropriate precautions that must be undertaken for successful experimentation.

<table><tbody><tr><td>Anti-BSA IgG1</td><td>Innovative Research</td><td>IBSA9E2C2</td><td></td></tr><tr><td>Alexa Fluor 647 Rat IgG2b, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400626</td><td></td></tr><tr><td>Anti-mouse CD16/32</td><td>BioLegend</td><td>101302</td><td></td></tr><tr><td>Anti-mouse F4/80 antibody conjugated to Alexa Fluor 647</td><td>BD Biosciences</td><td>565853</td><td></td></tr><tr><td>Anti-mouse F4/80 antibody conjugated to FITC</td><td>BioLegend</td><td>123108</td><td></td></tr><tr><td>Anti-mouse/human CD11b antibodyconjugated to Alexa Fluor 647&amp;nbsp;</td><td>BioLegend</td><td>101218</td><td></td></tr><tr><td>beta-mercaptoethanol (BME)</td><td>Sigma</td><td>M3148-100ml</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) FractionV</td><td>Fisher</td><td>BP1600-100</td><td></td></tr><tr><td>C57BL/6J&amp;nbsp;</td><td>Jackson labs</td><td>Stock No.000664</td><td></td></tr><tr><td>CM-H2DCFDA</td><td>Molecular Probes</td><td>C6827</td><td>Can be a substitute for oxidative stress detection reagent in the Enzo kit</td></tr><tr><td>Dihydroethidium (DHE)</td><td>Molecular Probes</td><td>D11347</td><td>Can be a substitute for superoxide detection reagent in the Enzo kit</td></tr><tr><td>DMEM 1x</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>DMEM no phenol red</td><td>Gibco</td><td>31053-028</td><td></td></tr><tr><td>DMF Anhydrous&amp;nbsp;</td><td>Acros Organics</td><td>61094-1000</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>FITC Rat IgG2a, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400506</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>L glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>LADMAC cells</td><td>ATCC</td><td>CRL-2420</td><td></td></tr><tr><td>MEM</td><td>Corning</td><td>10-010-CV</td><td></td></tr><tr><td>mouse IFN-g</td><td>GoldBio</td><td>1360-06-100</td><td></td></tr><tr><td>N-Acetyl-L-cysteine</td><td>EMD Milipore</td><td>106425</td><td>Can be a substitute for ROS inhibitor/scavenger in the Enzo kit</td></tr><tr><td>Novocyte flow cytometer with autosampler</td><td>Acea</td><td>2060R</td><td></td></tr><tr><td>Pyocyanin (ROS inducer)</td><td>Cayman chemical</td><td>10009594</td><td>Can be a substitute for inducer in the Enzo kit</td></tr><tr><td>ROS-ID total ROS/superoxide detection kit</td><td>ENZO</td><td>ENZ-51010</td><td></td></tr><tr><td>Sodium pyruvate (100mM)</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25200-056</td><td></td></tr></tbody></table>

flow cytometric measurement of ros production in macrophages in response to fc&#947;r cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

Using the protocol outlined within, we present representative data demonstrating flow cytometric detection of ROS production resulting from stimulation of WT C57BL/6J BMDMs through the Fc&#947;R. As expected, we observe minimal changes in FL1 or FL2 fluorescence above background levels in unstimulated cells (Figure 3A, compare &#8220;stained, unstimulated&#8221; vs &#8220;unstained, unstimulated&#8221; dot plots). We observe a marked increase in FL1 and FL2 fluorescence when cells are stimulated with Fc&#947;R cross-linking agent (Figure 3A, compare &#8220;stained and stimulated via Fc cross-linking&#8221; samples vs &#8220;stained, unstimulated&#8221; dot plots). Lastly, when cells were treated with ROS inhibitor prior to Fc&#947;R cross-linking, this increased fluorescence is brought back to basal levels (Figure 3A, compare &#8220;stained and treated with ROS inhibitor and stimulated via Fc cross-linking&#8221; vs &#8220;stained and stimulated via Fc cross-linking&#8221; dot plots). This is also evident when data is presented as a histogram for each channel (Figure 3B) or when data is presented as a percentage of cells positive for either the green or orange ROS probes (Figure 3C). A similar trend is also apparent when data is presented as MFI, although the reduction in orange fluorescence with pre-treatment with ROS inhibitor is not captured as well when presented as MFI versus as a percentage (Figure 3C). We also present the results of 3 independent experiments performed on different days (Figure 3, Experiment 1, 2, and 3). The average values and corresponding standard error of the mean are indicated in the graphs (Figure 3C).
We also present unsuccessful experimentation, where sub-optimal ROS production as a result of Fc&#947;R stimulation was observed (Figure 4). A minimal increase in FL1 and FL2 fluorescence was detected when comparing &#8220;stained and stimulated via Fc cross-linking&#8221; samples vs &#8220;stained, unstimulated&#8221; samples (Figure 4A,B,C). This is presented alongside a successful experiment to highlight the large differences between the expected percentages or MFI increases and the observed values in the unsuccessful experiment.
The current protocol utilizes a 24 h priming step. When comparing a 24 h versus a 48 h priming time, we observed no marked difference in the percentage of cells positive for the green, oxidative stress reagent (Figure 5A, top histograms and Figure 5B green probe, % positive). However, increasing the priming time to 48 h did increase the percentage of cells positive for the orange fluorescence (Figure 5A, lower histograms and Figure 5B orange probe, % positive). This was similarly reflected when data was presented as MFI. This suggests that for optimal detection of all ROS species, a 48 h priming time may be more ideal.
Given that, due to the cost or the time needed for experimentation, use of a kit to perform this assay may not be an option. For this reason, we also tested similar components to those provided in the kit and purchased these from standard vendors (Thermofisher, EMD Millipore, Cayman). We find that using individually procured components and the same experimental protocol for cell loading and Fc&#947;R stimulation, we can recapitulate many of the same findings we observed using the kit (Figure 6A,B). However, although increases in fluorescence were apparent with stimulation, a higher level of ROS production was observed using the kit. This may indicate that use of individually procured components may be feasible but would need to be further optimized for this specific assay.
Lastly, we demonstrate that it is also possible to combine cell-surface staining with these ROS probes. We use a known macrophage marker, F4/80, conjugated to Alexa 647 and perform cell surface staining prior to treatment with ROS inhibitor, ROS inducer, or specific stimuli to induce ROS production. We demonstrate in Figure 7B that macrophages respond as expected when treated with Fc&#947;R crosslinking agent and Fc&#947;R crosslinking agent + ROS inhibitor (Figure 7B, orange vs green dot plots). Furthermore, we can observe increased orange or green fluorescence specifically generated by the F4/80 labeled cells upon treatment with Fc&#947;R crosslinking agent and reduced with pre-treatment with ROS inhibitor (Figure 7B, F4/80 vs green and F4/80 vs orange dot plots).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59167/59167fig1.jpg" /> 
Figure 1: Flow cytometric assessment of appropriate generation of BMDMs. Wild type BMDMs were generated and were left either unstained or stained with FITC anti-mouse F4/80 or Alexa 647 anti-mouse CD11b. FSC (x-axis) vs SSC (y-axis) plots were generated and macrophages (BMDMs) were gated to exclude dead cells and debris. Using a plot of FSC-H(x-axis) vs FSC-A (y-axis), a singlet gate was generated. Gating on singlets, histograms were generated to show cells stained with either FITC F4/80 or APC CD11b in comparison to the isotype stained control. A) Correct BMDM differentiation with more than 95% of cells staining positive for CD11b or F4/80. B) Incorrect BMDM differentiation where less than 95% of cells are staining positive for F4/80 and 2 peaks are present for CD11b. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59167/59167fig2v2.jpg" /> 
Figure 2. Performing compensation when using green and orange ROS probes. Compensation will require unstained untreated cells, cells stained with green ROS probe and treated with ROS inducer, and cells stained with orange ROS probe and treated with ROS inducer. Dot plots for unstained untreated cells are used to determine quadrant gates. Data for cells singly stained with either green ROS probe or orange ROS probe and treated with ROS inducer are shown prior to, and after, compensation was applied. The compensation matrix is then applied to all subsequent experimental samples. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59167/59167fig3v2.jpg" /> 
Figure 3. Measurement of ROS in response to specific Fc&#947;R stimulation using green and orange ROS probes and assessment of assay reproducibility. Wild-type bone marrow-derived macrophages (BMDMs) were generated, primed, and were left either unstained or stained with a cocktail of green and orange ROS probes. Stained BMDMs were either left untreated, stimulated through their Fc&#947;Rs using murine anti-BSA IgG1 + BSA for 30 min, or treated with ROS inhibitor prior to stimulation via Fc&#947;R cross-linking. A) Dot plots, showing an increase in the percentages of cells in the upper left, upper right, and lower right quadrants upon specific Fc&#947;R stimulation, which is reduced in the presence of ROS inhibitor. B) Histograms of each fluorescence channel, showing the marker gate to determine cells positive for each probe. C) Presentation of the data as a percentage of cells positive for each probe or as an increase in MFI. Three independent, representative experiments are presented. The mean for the 3 experiments and standard errors of the mean are shown as lines within the graphs. Fc XL, Fc cross-linking; NAC, N-acetyl-L-cysteine. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59167/59167fig4v2.jpg" /> 
Figure 4. Examples of successful and suboptimal Fc&#947;R stimulation. Wild-type bone marrow-derived macrophages (BMDMs) were generated, primed, and were left either unstained or stained with a cocktail of green and orange ROS probes. Stained BMDMs were either left untreated, stimulated through their Fc&#947;Rs using murine anti-BSA IgG1 + BSA for 30 min, or treated with ROS inhibitor prior to stimulation via Fc&#947;R cross-linking. Representative results for successful and suboptimal stimulation are shown as A) dot plots, B) histograms, or C) the percentage of cells positive for each probe or as an increase in MFI. Successful stimulation shows increased fluorescence in the upper left, upper right and lower right quadrants of the FL1 vs FL2 plot and increased MFI and percentage of positive cells stained with each probe upon Fc&#947;R stimulation. Suboptimal stimulation shows minimal increase in MFI or percentage of positive cells. Fc XL, Fc cross-linking; NAC, N-acetyl-L-cysteine. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59167/59167fig5.jpg" /> 
Figure 5. Effect of priming time on ROS generation upon Fc&#947;R stimulation. BMDMs were generated, primed for either 24 or 48 h, and were left either unstained or stained with a cocktail of green and orange ROS probes. Stained BMDMs were either left untreated, stimulated through their Fc&#947;Rs using murine anti-BSA IgG1 + BSA for 30 min, or treated with ROS inhibitor prior to stimulation via Fc&#947;R cross-linking. A) Histograms for the fluorescence induced by each probe upon stimulation of macrophages primed for either 24 or 48 h. B) Percentage of cells positive for each probe upon stimulation of macrophages primed for either 24 or 48 h. Priming macrophages for 48 h resulted in an increase in percent of cells positive for orange fluorescence (or an increase in the MFI for the FL2 channel) compared to priming the macrophages for 24 h. The mean for the 3 experiments and standard errors of the mean are shown as lines within the graphs. Fc XL, Fc cross-linking; NAC, N-acetyl-L-cysteine. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59167/59167fig6v2.jpg" /> 
Figure 6. Flow cytometric ROS measurement upon Fc&#947;R cross-linking using reagents from different vendors. BMDMs were generated, primed for 24 h, and were left either unstained or stained with a cocktail of oxidative stress and superoxide detection probes. Stained BMDMs were either left untreated, stimulated with ROS inducer, stimulated through their Fc&#947;Rs using murine anti-BSA IgG1 + BSA for 30 min, or treated with ROS inhibitor prior to stimulation via Fc&#947;R cross-linking. Probes, ROS inducer and ROS inhibitor were used either from the kit or were purchased separately from different vendors and used at a similar concentration. A) Dot plots showing a side-by-side comparison of results obtained using kit and non-kit components. B) Histograms showing a side-by-side comparison of results obtained using kit and non-kit components. Fc XL, Fc cross-linking; NAC, N-acetyl-L-cysteine. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59167/59167fig7.jpg" /> 
Figure 7. Combining cell-surface staining with flow cytometric measurement of ROS production upon Fc&#947;R cross-linking. A) Dot plots for the various fluorescent channels using unstained or singly stained compensation controls. Wild-type BMDMs were generated, primed for 24 h, and were left either unstained, stained with Alexa 647 anti-mouse F4/80 only, stained with green ROS probe only and treated with ROS inducer, or stained with orange ROS probe only and treated with ROS inducer. Dot plots for unstained untreated cells are used to determine quadrant gates. Plots demonstrate expected results if channels are correctly compensated. The compensation matrix was then applied to all subsequent experimental samples. B) Wild-type BMDMs were generated, primed for 24 h and stained with Alexa 647 anti-mouse F4/80. Afterward, BMDMs were either left unstimulated, stimulated through their Fc&#947;Rs using murine anti-BSA IgG1 + BSA for 30 min, or treated with ROS inhibitor prior to stimulation via Fc&#947;R cross-linking. Dot plots demonstrate that green or orange fluorescence specifically produced by F4/80 cells can be detected. <a href="https://www.jove.com/files/ftp_upload/59167/59167fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

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15.8K Views.  University of Central Florida. This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the FcγR. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct FcγR cross-linking.

Video: Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Medicine

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Biochemistry

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Biology

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Neuroscience

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Detecting Reactive Oxygen Species

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are many deleterious effects associated with unchecked ROS production, including structural damage to DNA and other biological molecules. However, ROS can also be mediators of physiological signaling. There is accumulating evidence that ROS play significant roles in everything from activation of transcription factors to the mediation of inflammatory toxicity that kills foreign pathogens and defend the body.In this video we will delve into the associations between ROS, metabolism and disease. After establishing their significance, we will discuss the principles and a protocol of a commonly used methodology for measuring ROS levels in cells: the use of non-fluorescent probes that become fluorescent upon oxidation. Lastly, we will review some current applications of this technique in cell biology research.

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species are chemically active, oxygen-derived molecules capable of oxidizing other molecules. Because of their reactive nature, there are ...

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Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app.jove.com/v/63162">Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.</a> J. Vis. Exp. (2022).
This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

Source: Bol&#237;var, B. E., et. al., Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages. J. Vis. Exp. 
...

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Immunology

Immune Response

Immune Modulation

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://app.jove.com/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

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