The authors would like to acknowledge past and present members of the Kim laboratory, Mark Nilan for zebrafish care and maintenance, Dr. Robert Wheeler for helpful discussions and data sharing, and NIH grants 3RO1GM087308-02S1 and 1P20RR024475-01A2 and the Maine Agricultural and Forest Experiment Station (Publication Number 3303) for funding.

1. Zebrafish Care and Maintenance
<ol>
	<li>Husbandry: Mass spawn adult zebrafish as previously described13. Collect spawned embryos as previously described14.</li>
	<li>Microinjection (if desired): Microinject 1-4 cell stage zebrafish embryos with morpholino oligonucleotides to knockdown gene products or mRNA to overexpress gene products as previously described15.
	<ol>
		<li>Maintain an adequate pool of mock injected controls (at least 48 living, mock injected control fish and 48 livi...

<ol>
	<li>Medzhitov, R., Janeway, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+Immunity:+Impact+on+the+Adaptive+Immune+Response.">Innate Immunity: Impact on the Adaptive Immune Response.</a> Current Opinion in Immunology. 9, 4-9 (1997).</li><li>Lam, S. H., Chua, H. L., 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=Development+and+Maturation+of+the+Immune+System+in+Zebrafish,+Danio+rerio:+A+Gene+expression+Profiling.">Development and Maturation of the Immune System in Zebrafish, Danio rerio: A Gene expression Profiling.</a> In Situ Hybridization and Immunological. 28, 9-28 (2004).</li><li>Stockhammer, O. W., Zakrzewska, 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=Transcriptome+Profiling+and+Functional+Analyses+of+the+Zebrafish+Embryonic+Innate+Immune+Response+to+Salmonella+Infection.">Transcriptome Profiling and Functional Analyses of the Zebrafish Embryonic Innate Immune Response to Salmonella Infection.</a> J Immunol. 9. 9, 5641-5653 (2009).</li><li>Ordas, A., Hegedus, Z., 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=Deep+Sequencing+of+the+Innate+Immune+Transcriptomic+Response+of+Zebrafish+Embryos+to+Salmonella+Infection.">Deep Sequencing of the Innate Immune Transcriptomic Response of Zebrafish Embryos to Salmonella Infection.</a> Fish &amp; Shellfish Immunology. 31, 716-724 (2011).</li><li>Renshaw, S. A., Loynes, C. 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=A+Transgenic+Zebrafish+Model+of+Neutrophilic+Inflammation.">A Transgenic Zebrafish Model of Neutrophilic Inflammation.</a> Blood. 13, 3976-3978 (2006).</li><li>Mathias, J. R., Perrin, B. J., 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=Resolution+of+Inflammation+by+Retrograde+Chemotaxis+of+Neutrophils+in+Transgenic+Zebrafish.">Resolution of Inflammation by Retrograde Chemotaxis of Neutrophils in Transgenic Zebrafish.</a> J. Leukoc. Biol. 6, 1281-1288 (2006).</li><li>Ellett, F., Pase, L., 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=mpeg1+Promoter+Transgenes+Direct+Macrophage-Lineage+Expression+in+Zebrafish.">mpeg1 Promoter Transgenes Direct Macrophage-Lineage Expression in Zebrafish.</a> Blood. 4, 56-56 (2011).</li><li>Phennicie, R. T., Sullivan, M. J., 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=Specific+Resistance+to+Pseudomonas+aeruginosa+Infection+in+Zebrafish+is+Mediated+by+the+Cystic+Fibrosis+Transmembrane+Conductance+Regulator.">Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator.</a> Infect. Immun. 11, 4542 (2010).</li><li>Brothers, K. M., Newman, Z. R., 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=Live+Imaging+of+Disseminated+Candidiasis+in+Zebrafish+Reveals+Role+of+Phagocyte+Oxidase+in+Limiting+Filamentous+Growth.">Live Imaging of Disseminated Candidiasis in Zebrafish Reveals Role of Phagocyte Oxidase in Limiting Filamentous Growth.</a> Eukaryotic Cell. 7, 932-944 (2011).</li><li>Rotman, J., van Gils, W., 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=Rapid+Screening+of+Innate+Immune+Gene+Expression+in+Zebrafish+using+Reverse+Transcription+-+Multiplex+Ligation-Dependent+Probe+Amplification.">Rapid Screening of Innate Immune Gene Expression in Zebrafish using Reverse Transcription - Multiplex Ligation-Dependent Probe Amplification.</a> BMC Research Notes. 4, (2011).</li><li>d'Alencon, C. A., Pena, O. 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=A+High-Throughput+Chemically+Induced+Inflammation+Assay+in+Zebrafish.">A High-Throughput Chemically Induced Inflammation Assay in Zebrafish.</a> BMC Biology. 8, 151 (2010).</li><li>Hermann, A. C., Millard, P. J., 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=Development+of+a+Respiratory+Burst+Assay+using+Zebrafish+Kidneys+and+Embryos.">Development of a Respiratory Burst Assay using Zebrafish Kidneys and Embryos.</a> Journal of Immunological Methods. 292, 119-129 (2004).</li><li>Avdesh, A., Chen, M., 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=Regular+Care+and+Maintenance+of+a+Zebrafish+(Danio+rerio)+Laboratory:+An+Introduction.">Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction.</a> J. Vis. Exp. (69), e4196 (2012).</li><li>Brothers, K. M., Wheeler, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Imaging+of+Disseminated+Candidiasis+in+Zebrafish+Larvae.">Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae.</a> J. Vis. Exp. (65), e4051 (2012).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Gerlach, G. F., Schrader, L. N., 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=Dissection+of+the+Adult+Zebrafish+Kidney.">Dissection of the Adult Zebrafish Kidney.</a> J. Vis. Exp. (54), e2839 (2011).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Organs+from+the+Adult+Zebrafish.">Dissection of Organs from the Adult Zebrafish.</a> J. Vis. Exp. (37), e1717 (2010).</li><li>Le Guyader, D., Redd, M. J., 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=Origins+and+Unconventional+Behavior+of+Neutrophils+in+Developing+Zebrafish.">Origins and Unconventional Behavior of Neutrophils in Developing Zebrafish.</a> Blood. 111, 132-141 (2008).</li><li>Davidson, A. J., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'Definitive'+(and+'Primitive')+Guide+to+Zebrafish+Hematopoiesis.">The 'Definitive' (and 'Primitive') Guide to Zebrafish Hematopoiesis.</a> Oncogene. 23, 7233-7246 (2004).</li><li>Jovanovic, B., Goetz, F. W., 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=Immunological+Stimuli+Change+Expression+of+Genes+and+Neutrophil+Function+in+Fathead+Minnow+Pimephales+promelas+Rafinesque.">Immunological Stimuli Change Expression of Genes and Neutrophil Function in Fathead Minnow Pimephales promelas Rafinesque.</a> Journal of Fish Biology. 78, 1054-1072 (2011).</li><li>Niethammer, P., Grabher, C., 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=A+Tissue-Scale+Gradient+of+Hydrogen+Peroxide+Mediates+Rapid+Wound+Detection+in+Zebrafish.">A Tissue-Scale Gradient of Hydrogen Peroxide Mediates Rapid Wound Detection in Zebrafish.</a> Nature. 459, 996-1000 (2009).</li><li>Thisse, B., Pflumio, S., 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=Expression+of+the+zebrafish+genome+during+embryogenesis.+(NIH+R01+RR15402).">Expression of the zebrafish genome during embryogenesis. (NIH R01 RR15402).</a> ZFIN Direct Data Submission. , (2001).</li><li>Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Release+Clones:+A+High+Throughput+Expression+Analysis.">Fast Release Clones: A High Throughput Expression Analysis.</a> ZFIN Direct Data Submission. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Table+18.4.">Table 18.4.</a> The Molecular Probes Handbook. , .</li></ol>

The primary function of phagocytes is to detect, engulf, and destroy pathogens. The ability of phagocytes to produce an adequate respiratory burst is critical for this function. Thus, quantification of the respiratory burst response is one method to allow comparison of general innate immune health and function between groups of individuals and/or in response to experimental manipulations. Here, we describe a protocol to induce, quantify, and compare the respiratory burst response between groups of individual zebraf...

The immune system is comprised of two branches: innate and adaptive immunity. Innate immunity is evolutionarily more ancient than adaptive immunity. Invertebrates are presently thought to have only innate immunity, whereas vertebrates possess both the innate and adaptive branches. While adaptive immunity confers specific and long-lasting immunity to certain pathogens, innate immunity is an immediate response to invading bacteria, viruses, and fungi. A crucial aspect of the innate immune response involves the release of cytokines and chemokines, which results in inflammation and recruitment of phagocytes (e.g. macrophages, neutrophils) to engulf and destroy foreign invaders.
Successful innate immune responses involve: (1) recognition of invading microorganisms; (2) induction of the appropriate signaling cascades (e.g. release of cytokines and chemokines); (3) proper development/adequate numbers of phagocytic cells; (4) migration of phagocytes to sites of infection; (5) engulfment of pathogens; and (6) destruction of engulfed microorganisms. A deficiency in any one of these steps could lead to the host being overwhelmed by, and succumbing to, the infection. A robust innate immune response is vital to the health of organisms because it is the first line of defense against pathogens in all plants and animals. In vertebrates, it also potentiates the adaptive immune response1. Therefore, it is critical that we are able to evaluate all aspects of the innate immune response in order to better understand it and to optimize its function.
Many model organisms are used to study innate immunity, ranging from Arabadopsis to C. elegans to Drosophila to mice to cultured human cells. An advantage to using the zebrafish (Danio rerio) model system to study innate immunity is that the zebrafish is a vertebrate, with both innate and adaptive immunity, yet the development of innate and adaptive immunity are temporally segregated. Zebrafish rely solely on innate immunity for protection against infection until adaptive immunity becomes fully functional, which occurs around 4-6 weeks post fertilization2. In addition to tools for genetic manipulation, optical clarity and rapid, external development, innate immunity as the principle mode of defense in zebrafish embryos provides a simplified model in which to study the complexities of the innate immune response in vivo.
Multiple protocols have been developed to assess different facets of the innate immune response in zebrafish embryos. Microarrays and RNAseq have validated that the cytokine profiles elicited by the zebrafish innate immune response are similar to that of humans and have also suggested the involvement of unexpected genes in innate immunity3,4. The transparency of the zebrafish embryo and fluorescent, transgenic strains of pathogens and zebrafish allow for visualization of dynamic host-pathogen interactions in vivo in real time. Transgenic zebrafish embryos expressing GFP under control of the neutrophil-specific myeloperoxidase promoter5,6 or the macrophage-specific mpeg1 promoter7 have made it possible to visualize and quantify phagocyte migration to sites of localized infections8 as well as to visualize phagocytosis and destruction of fluorescently labeled pathogens8,9. Zebrafish embryos are also amenable to the generation of high-throughput assays and chemical screens. Accordingly, high-throughput methods of transcriptome analysis upon infection10 and phagocyte migration to sites of chemically induced injury11 have recently been developed.
Of the techniques listed above, none quantitatively assess the final stage of pathogen destruction by phagocytes. This final stage involves a respiratory burst (i.e. production of ROS and other toxic compounds), which kill the engulfed pathogens. The enzyme NADPH oxidase is a major source of ROS in phagocytic cells. Assembly of the subunits of the NADPH oxidase enzyme results in transfer of electrons to oxygen, generating superoxide anions. Through subsequent enzymatic reactions, superoxide can then be converted into hydrogen peroxide and hypochlorous acid (Figure 1A). It is the respiratory burst of phagocytes that kills pathogens and thus, the quantification of the respiratory burst potential of zebrafish embryos is indicative of overall innate immune health. We developed a fluorescence-based assay to quantify the respiratory burst in groups of individual zebrafish embryos12. This assay utilizes the non-fluorescent, reduced form of a commercially available, cell-permeable dye. This dye, 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (H2DCFDA), is converted into the fluorescent compound, 2&#39;,7&#39;-dichlorofluorescein (DCF), upon oxidation. The diverse ROS generated by the phagocyte respiratory burst can oxidize H2DCFDA and generate fluorescence24. The appearance of fluorescence can be used to quantify and compare the respiratory burst response between groups of zebrafish. The protein kinase C agonist phorbol myristate acetate (PMA) is used to chemically induce NADPH oxidase to produce ROS and thus increase fluorescence readings (Figure 1B). Herein, we provide a detailed protocol of a modified and optimized version of this zebrafish embryo respiratory burst assay. This assay can be used to compare the respiratory burst between groups of individual zebrafish embryos over time and/or in response to experimental manipulations (e.g. morpholino-mediated protein knockdown). The use of this method, in conjunction with other zebrafish innate immunity assays, will provide a more complete picture of the complex and critical innate immune response.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Instant Ocean Sea Salt</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>H2DCFDA</td>
			<td>Sigma Aldrich</td>
			<td>35845-1G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>PMA</td>
			<td>Fisher</td>
			<td>BP6851</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2438-5X10ML</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tricaine S MS222</td>
			<td>Western Chemical</td>
			<td>100 grams</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM/F-12, No Phenol Red </td>
			<td>Life Technologies</td>
			<td>11039-021</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Deep Petri Dishes</td>
			<td>VWR</td>
			<td>89107-632</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plastic Transfer Pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>#5 Dumont Forceps</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1.7 ml Micro Centrifuge Tubes</td>
			<td>Axygen</td>
			<td>10011-724</td>
			<td></td>
		 </tr>
		 <tr>
			<td>15 ml Conical Centrifuge Tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml Serological Pipettes</td>
			<td>Greiner Bio One</td>
			<td>606180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synergy 2 Multi-Mode Microplate Reader</td>
			<td>BioTek</td>
			<td>Contact BioTek</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Black 96 Well Microplate</td>
			<td>VWR</td>
			<td>82050-728</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml Sterile Reservoirs</td>
			<td>VistaLab</td>
			<td>3054-2003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>P200 Pipettor</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Multichannel Pipettor</td>
			<td>VWR</td>
			<td>89079-948</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipette Tips</td>
			<td>VWR</td>
			<td>89079-478</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

quantification of the respiratory burst response as an indicator of innate immune health in zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

Here, we provide data comparing the respiratory burst response in zebrafish embryos (wild-type, AB background) at 48 and 72 hours post fertilization (hpf). The 48 hpf embryos acted as our control group and the 72 hpf embryos as our experimental group. The sample size used was 24 un-induced embryos and 24 PMA-induced embryos per developmental stage. Raw fluorescence readings (in Relative Fluorescence Units (RFU)) were obtained by reading the microplate 4 hours after the addition of PMA. Raw fluorescence values are provide...

Watch this Scientific Journal Video about Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish at JoVE.com

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

quantification-respiratory-burst-response-as-an-indicator-innate

Research

JoVE Journal

Immunology and Infection

21.5K vistas.  University of Maine.  La respuesta inmune innata protege a los organismos contra la infección por patógenos. Un componente fundamental de la respuesta inmune innata, el estallido respiratorio de fagocitos, genera especies reactivas del oxígeno que matan los microorganismos invasores. Se describe un ensayo de estallido respiratorio que cuantifica las especies reactivas del oxígeno producido cuando la respuesta inmune innata es inducida químicamente. 