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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+Oxygen+Species+and+Neutrophil+Function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+function+in+redox+signaling+and+oxidative+stress.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+phagocytic+leukocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phagocyte+respiratory+burst:+Historical+perspectives+and+recent+advances.">The phagocyte respiratory burst: Historical perspectives and recent advances.</a> Immunology Letters. 192, 88-96 (2017).</li><li>Fang, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+actions+of+reactive+oxygen+species.">Antimicrobial actions of reactive oxygen species.</a> MBio. 2 (5), (2011).</li><li>Iles, K. E., Forman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+signaling+and+respiratory+burst.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+superoxide+production+by+granulocytes+from+patients+with+chronic+granulomatous+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatal+(chronic)+granulomatous+disease+of+childhood:+a+hereditary+defect+of+leukocyte+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+metabolic+activity+of+leukocytes+from+patients+with+a+genetic+abnormality+of+phagocytic+function.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pattern+of+genetic+transmission+of+the+leukocyte+defect+in+fatal+granulomatous+disease+of+childhood.">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><li>Dikalov, S. I., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+detection+of+mitochondrial+and+cellular+reactive+oxygen+species.">Methods for detection of mitochondrial and cellular reactive oxygen species.</a> Antioxidants &amp; Redox Signaling. 20 (2), 372-382 (2014).</li><li>Held, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Reactive+Oxygen+Species:+Measurement+of+ROS+in+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+reactive+oxygen+species+measurement+in+biological+systems.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2',7'-Dichlorodihydrofluorescein+as+a+fluorescent+probe+for+reactive+oxygen+species+measurement:+Forty+years+of+application+and+controversy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroethidine-+and+MitoSOX-derived+red+fluorescence+is+not+a+reliable+indicator+of+intracellular+superoxide+formation:+another+inconvenient+truth.">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><li>Swamydas, M., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+purification+and+labeling+of+mouse+bone+marrow+neutrophils+for+functional+studies+and+adoptive+transfer+experiments.">Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments.</a> Journal of Visualized Experiments. (77), e50586 (2013).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" style="width:623px;" width="623">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-BSA IgG1</td>
			<td style="width:121px;">Innovative Research</td>
			<td style="width:119px;">IBSA9E2C2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa Fluor 647 Rat IgG2b, &#954; Isotype Ctrl Antibody</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:119px;">400626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-mouse CD16/32</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:119px;">101302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-mouse F4/80 antibody conjugated to Alexa Fluor 647</td>
			<td style="width:121px;">BD Biosciences</td>
			<td style="width:119px;">565853</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-mouse F4/80 antibody conjugated to FITC</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:119px;">123108</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse/human CD11b antibodyconjugated to Alexa Fluor 647&#160;</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:119px;">101218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">beta-mercaptoethanol (BME)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M3148-100ml</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bovine Serum Albumin (BSA) FractionV</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:119px;">BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57BL/6J&#160;</td>
			<td style="width:121px;">Jackson labs</td>
			<td>Stock No.000664</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CM-H2DCFDA</td>
			<td style="width:121px;">Molecular Probes</td>
			<td style="width:119px;">C6827</td>
			<td>Can be a substitute for oxidative stress detection reagent in the Enzo kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dihydroethidium (DHE)</td>
			<td style="width:121px;">Molecular Probes</td>
			<td style="width:119px;">D11347</td>
			<td>Can be a substitute for superoxide detection reagent in the Enzo kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM 1x</td>
			<td style="width:121px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM no phenol red</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">31053-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMF Anhydrous&#160;</td>
			<td style="width:121px;">Acros Organics</td>
			<td style="width:119px;">61094-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:121px;">VWR</td>
			<td style="width:119px;">97068-085</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FITC Rat IgG2a, &#954; Isotype Ctrl Antibody</td>
			<td style="width:121px;">BioLegend</td>
			<td style="width:119px;">400506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES (1M)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L glutamine</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LADMAC cells</td>
			<td style="width:121px;">ATCC</td>
			<td>CRL-2420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM</td>
			<td style="width:121px;">Corning</td>
			<td style="width:119px;">10-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse IFN-g</td>
			<td style="width:121px;">GoldBio</td>
			<td style="width:119px;">1360-06-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-Acetyl-L-cysteine</td>
			<td style="width:121px;">EMD Milipore</td>
			<td style="width:119px;">106425</td>
			<td>Can be a substitute for ROS inhibitor/scavenger in the Enzo kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Novocyte flow cytometer with autosampler</td>
			<td style="width:121px;">Acea</td>
			<td style="width:119px;">2060R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pyocyanin (ROS inducer)</td>
			<td style="width:121px;">Cayman chemical</td>
			<td style="width:119px;">10009594</td>
			<td>Can be a substitute for inducer in the Enzo kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ROS-ID total ROS/superoxide detection kit</td>
			<td style="width:121px;">ENZO</td>
			<td style="width:119px;">ENZ-51010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate (100mM)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">11360-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/59167/59167fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÎ³R Cross-linking at JoVE.com

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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Nanyang Technological University

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JoVE Journal

Immunology and Infection

15.6K 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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

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

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Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro