Name: flow cytometric measurement of ros production in macrophages in response to fc&#947;r cross-linking
Uploaded: 2019-03-07T15:30:00
Description: Watch this Scientific Journal Video about Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃŽÂ³R Cross-linking at JoVE.com

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.

The protocol for animal handling was approved by the Institutional Animal Care and Use committee (IACUC) of University of Central Florida.
1. Generation of bone marrow derived macrophages (BMDMs)
<ol>
	<li>Culture media preparation
	<ol>
		<li>Prepare D10F base media: To Dulbecco&#8217;s Modified Eagle Medium (DMEM), add 10% heat inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.05 mM &#946;-me...

<ol>
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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 th...

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 stimul...

Watch this Scientific Journal Video about Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃŽÂ³R Cross-linking at JoVE.com

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R 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.

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 ...

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