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 living, experimentally manipulated fish are needed to fill a 96 well microplate).</li>
	</ol>
	</li>
	<li>Maintain embryos: Grow embryos in deep petri dishes at 28 &#176;C in egg water (60 &#956;g/ml Instant Ocean Sea Salt in distilled water-autoclaved) until the desired developmental stage (a respiratory burst response is not detectable using this protocol in zebrafish embryos younger than 2 days post fertilization12). Note: It has been observed that preventing pigmentation in zebrafish embryos through either the use of 1-phenyl 2-thiourea (PTU) or golden/slc24a5 mutant zebrafish does not significantly alter the induction of fluorescence by PMA (unpublished data, embryos tested at 48, 72, and 96 hpf).
	<ol>
		<li>Remove dead embryos daily with a plastic transfer pipette.</li>
		<li>Carefully decant old egg water and replenish with new egg water daily.</li>
	</ol>
	</li>
	<li>Dechorionate embryos: On the day of the experiment, dechorionate embryos (if embryos are still in their chorions) as previously described using two fine forceps14 (this respiratory burst assay can also be performed on dissected kidneys from adult zebrafish12 and detailed protocols for kidney dissection from adult zebrafish have been previously described16,17).</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare a stock solution of H2DCFDA: Weigh out 1 mg of H2DCFDA.
	<ol>
		<li>Dissolve 1 mg of H2DCFDA in 1 ml of dimethyl sulfoxide (DMSO) to make a 1 mg/ml stock solution.</li>
		<li>Make 22 &#956;l aliquots of this stock solution in 1.7 ml microcentrifuge tubes.</li>
		<li>Wrap the aliquots of H2DCFDA stock solution in aluminum foil and keep in the dark whenever possible because H2DCFDA is light sensitive.</li>
		<li>Store H2DCFDA aliquots at -20 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>CAUTION - Prepare a stock solution of phorbol myristate acetate (PMA): Weigh out 1 mg of PMA using proper personal protective equipment (i.e. gloves, goggles and mask).
	<ol>
		<li>Dissolve PMA in 1 ml of DMSO to make a 1 mg/ml stock solution.</li>
		<li>Make 11 &#956;l aliquots in 1.7 ml microcentrifuge tubes.</li>
		<li>Store aliquots of PMA stock solution at -80 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>Prepare a working solution of H2DCFDA: On the day of the experiment, make a H2DCFDA working solution by adding 1 part H2DCFDA stock solution to 1 part DMSO (500 &#956;g/ml H2DCFDA final concentration). For example, add 20 &#956;l of H2DCFDA stock solution and 20 &#956;l DMSO to a foil wrapped 1.7 ml microcentrifuge tube.</li>
	<li>Prepare a working solution of PMA: On the day of the experiment, make a PMA working solution by adding 1 part PMA stock solution to 49 parts nuclease free water (20 &#956;g/ml PMA final concentration). For example, dilute 10 &#956;l PMA stock solution in 490 &#956;l nuclease free water in a 1.7 ml microcentrifuge tube.</li>
	<li>Prepare a dosing solution of H2DCFDA: On the day of the experiment, make a H2DCFDA dosing solution with a final concentration of 1 &#956;g/ml H2DCFDA in egg water. The prepared volumes of the dosing solutions can be modified as desired, but ensure that the final concentrations of the reagents are maintained. For kidneys, instead of egg water, use the same volume of Dulbecco&#39;s modified Eagle&#39;s medium/F-12 (50% DMEM, 50% F-12, without phenol red). To make 5 ml of H2DCFDA dosing solution, use a 5 ml serological pipette to transfer 5 ml of egg water (or DMEM/F-12) into a 15 ml conical centrifuge tube wrapped in foil and labeled &#39;H&#39;.
	<ol>
		<li>Remove 10 &#956;l of egg water (or DMEM/F-12) from the 15 ml conical tube labeled &#39;H&#39; and discard.</li>
		<li>Add 10 &#956;l of H2DCFDA working solution into the 15 ml conical tube labeled &#39;H&#39; and vortex to mix (this volume is enough for 48 embryo or kidney samples (half of a full 96 well microplate) and these wells will provide measurements of the level of background fluorescence).</li>
	</ol>
	</li>
	<li>Prepare a dosing solution of H2DCFDA + PMA: On the day of the experiment, make a H2DCFDA + PMA dosing solution with final concentrations of: H2DCFDA - 1 &#956;g/ml and PMA - 400 ng/ml. To make 5 ml of H2DCFDA + PMA dosing solution, use a 5 ml serological pipette to transfer 5 ml of egg water (or DMEM/F-12) into a new 15 ml conical tube labeled &#39;H + P&#39;.
	<ol>
		<li>Remove 110 &#956;l of egg water (or DMEM/F-12) from the 15 ml conical tube labeled &#39;H + P&#39; and discard.</li>
		<li>Add 10 &#956;l of H2DCFDA working solution, then add 100 &#956;l of PMA working solution into the 15 ml conical tube labeled &#39;H + P&#39; and vortex to mix (this volume is enough for 48 samples or the other half of a full 96 well microplate).</li>
	</ol>
	</li>
	<li>Keep the dosing solutions on ice.</li>
</ol>
3. Microplate Reader Programming
<ol>
	<li>Prepare instrument: Power on the microplate reader.
	<ol>
		<li>Warm up the light source.</li>
		<li>Set up a program to read fluorescence: e.g. Excitation: 485 nm; Emission: 528 nm; Optics Position: top 510 nm; Sensitivity: 65, with a 5 sec shaking step prior to the read.</li>
	</ol>
	</li>
</ol>
4. 96 Well Microplate Set Up (see Figure 2)
<ol>
	<li>Gather supplies: Obtain dishes with dechorionated embryos, black 96 well microplate, p200 pipettor and tips, ice bucket with H2DCFDA and H2DCFDA + PMA dosing solutions, multichannel p200 pipettor, two sterile reservoirs, aluminum foil, and scissors.</li>
	<li>Transfer one embryo into each well of a 96 well microplate: Use scissors to cut a pipette tip such that embryos or kidneys fit through the opening.
	<ol>
		<li>Set a p200 pipettor to 100 &#956;l and transfer one embryo along with egg water (or DMEM/F-12) into as many of the wells of a black 96 well microplate as desired (it is not necessary to change the pipette tip for each different embryo sample within an experimental condition, but it may be necessary to change tips between experimental conditions). Be sure to avoid transferring residual chorions, as these tend to skew the data collected. For the transfer of kidneys, a larger volume pipettor (set to 100 &#956;l) can be used and pipette tips can be cut to obtain a larger bore size, if necessary. It may be necessary to incorporate wells without embryo or kidney samples, but with the dosing solutions to control for some experimental manipulations.</li>
	</ol>
	</li>
	<li>Add dosing solutions: Pour H2DCFDA dosing solution into a sterile 25 ml reservoir.
	<ol>
		<li>Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 &#956;l of H2DCFDA dosing solution into one column on the 96 well microplate (500 ng/ml final concentration of H2DCFDA).</li>
		<li>Repeat this (changing tips is not necessary) for as many columns as desired (usually six columns or 48 wells if filling an entire 96 well microplate). Add this solution to half of the control embryo samples and half of the experimentally manipulated embryo samples (an example 96 well microplate set up is shown in Figure 2, these wells (colored orange) will provide background fluorescence data in samples not induced with PMA).</li>
		<li>Pour the H2DCFDA + PMA dosing solution into a new, sterile 25 ml reservoir.</li>
		<li>Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 &#956;l of H2DCFDA + PMA dosing solution into the remaining columns (colored red in Figure 2) of the 96 well microplate (changing tips is not necessary, final concentrations of H2DCFDA- 500 ng/ml and PMA- 200 ng/ml).</li>
	</ol>
	</li>
	<li>Cover the microplate with aluminum foil.</li>
	<li>Shake the microplate for approximately 20 sec at 150 rpm to homogenize the solutions in each well.</li>
	<li>Incubate the microplate at 28 &#176;C when it is not being read.</li>
</ol>
5. Fluorescence Quantification
<ol>
	<li>Read the microplate at time = 0 hr after the addition of PMA using the parameters described in step 3.1.2.
	<ol>
		<li>Continue taking measurements every few minutes for the desired time interval or incubate the foil wrapped microplate at 28 &#176;C until a later time point and then take an endpoint measurement at a desired time (e.g. 4 hr after the addition of PMA).</li>
	</ol>
	</li>
	<li>Use a plastic transfer pipette to retrieve the embryos from the wells.</li>
	<li>Euthanize the embryos according to your animal care and usage protocol, e.g. immersion in tricaine MS222.</li>
	<li>Dispose of the microplate and other disposable materials in the biohazardous waste container.</li>
</ol>
6. Data Analysis
<ol>
	<li>Decide on the time point at which you would like to compare fluorescence values (e.g. 4 hr after the addition of PMA, Table 1).</li>
	<li>Subtract the average un-induced control group&#39;s fluorescence value from the individual PMA-induced control group&#39;s fluorescence values.</li>
	<li>Repeat this for the experimental group with and without PMA.</li>
	<li>Store these normalized fluorescence values in two columns, the control + PMA group and the experimental + PMA group (Table 2).</li>
	<li>Calculate the means and standard deviations for the normalized fluorescence values of the control + PMA group and the experimental + PMA group.</li>
	<li>Compare the normalized fluorescence values using an unpaired t-test to determine statistical significance (Table 2).</li>
	<li>Graph the means of the control + PMA group and the experimental + PMA group with error bars reflecting the appropriate standard deviations.</li>
	<li>Record the level of significance on the graph and in the figure legend (Figure 2).</li>
</ol>

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The authors have nothing to disclose.

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><tbody><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

21.6K Views.  University of Maine. 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.

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

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Neuroscience

Novel Tank Diving Assay: A Method to Study Innate Stress Responses in Zebrafish

Source: Chin J.S.R., et al. <a href="https://www.jove.com/v/59092/behavioral-approaches-to-studying-innate-stress-in-zebrafish" target="_blank">Behavioral Approaches to Studying Innate Stress in Zebrafish</a>. J. Vis. Exp. (2019)
The article describes a behavioral method to assess stress in zebrafish. In response to stress, zebrafish tends to swim at bottom of a tank until stress is removed. The sample protocol describes stepwise method for performing the assay.

Source: Chin J.S.R., et al. Behavioral Approaches to Studying Innate Stress in Zebrafish. J. Vis. Exp. (2019)
The article describes a behavioral method to ...

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

Summary

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Chapters in this video

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Novel Tank Diving Assay: A Method to Study Innate Stress Responses in Zebrafish