Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

1. Maintenance of Macrophage Cell Lines
<ol>
	<li>Grow RAW264.7 and J774A.1 cell lines in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 &#186;C in the presence of 5% CO2. To help maintain sterility, add 1% penicillin/streptomycin (pen/strep) to the media (although this is not strictly necessary).</li>
	<li>Critical step: Ensure that the macrophages are growing in a healthy state for efficient transfection and siRNA-induced gene knockdown. Use cells between passages 3 and 10 after fresh thawing, as these macrophage lines exhibit the best transfection efficiency and maintain robust post-transfection survival at this stage; do not use a higher passage number (&#62;20), as transfection efficiency and gene knockdown both diminish greatly.</li>
	<li>For optimized siRNA transfection, plate cells at 20% confluency and grow one or two days until the cells reach no more than 80% confluence. Overgrown cells will not transfect efficiently.</li>
</ol>
2. Programming the 96-well Shuttle System for Transfection
<ol>
	<li>Start the 96-well shuttle software on the computer connected to the shuttle. Select new parameter file from the top left file menu.</li>
	<li>Highlight the wells that will be transfected on the displayed 96-well plate schematic. A green box will indicate which wells are selected.</li>
	<li>Select the program that corresponds to the macrophage cell line being used (DS-136 for RAW264.7 or DS-130 for J774A.1) and select apply, located below the diagramed 96-well plate. Note that after selecting a program, the wells on the schematic plate will be colored dark blue. Empty circles indicate wells that have not been assigned a program and will not be shocked.</li>
</ol>
3. Preparing the Reagents for Transfection
<ol>
	<li>Prepare the working Nucleofector&#8482; solution by mixing SF cell line 96-well Nucleofector&#8482; solution with the entire contents of the enclosed supplement tube. Allow the mixed reagents to equilibrate at room temperature prior to use. Store excess complete Nucleofector&#8482; solution at 4 &#186;C for up to 3 months for future use.</li>
	<li>Pre-warm the DMEM, RPMI-1640, 0.25% trypsin/EDTA, and phosphate-buffered saline (PBS) at 37 &#186;C prior to preparing the macrophages for transfection.</li>
	<li>Prepare the siRNA for transfection by thawing on ice. In order to minimize the number of freeze thaws of the siRNA, freeze new siRNAs in aliquots the first time they are used.</li>
	<li>In initial experiments, use siRNA at a relatively high dose of 2 &#181;M (diluted 1:10 from 20 &#181;M stocks). Subsequently titrate siRNAs that induce robust gene knockdown down to lower doses, although typically higher doses are needed for efficient gene knockdown in macrophages compared to other cell types. 
	NOTE: siRNAs are available from many sources; fairly robust gene knockdown can be obtained using siGenome SMARTPOOLs, which are pools of four siRNAs targeting each gene. As a negative control, use any of various non-targeting siRNA pools or siRNAs.</li>
	<li>To monitor transfection efficiency, transfect either the pmaxGFP control plasmid (provided in the Amaxa kits) or fluorescently-labeled siRNAs in parallel to the experimental siRNAs. Expect 30-50% transfection of the GFP plasmid (assayed by microscopy or flow cytometry the day after transfection) and &#62;99% transfection of fluorescently-labeled siRNA (assayed by flow cytometry immediately after transfection and recovery).</li>
</ol>
4. Preparing the Macrophages for Transfection
<ol>
	<li>Detach adherent macrophages from the cell culture dishes by trypsinization. Aspirate the media and wash the cells twice with 10 ml PBS. After removing the PBS, incubate the cells with pre-warmed 0.25% trypsin/EDTA (1 ml/10 cm plate) for 1-2 min until cells begin to detach. Gently tap the plate and move the solution during the incubation to maximize trypsinization.</li>
	<li>After the incubation, add 9 ml DMEM + FBS media, mix gently up and down, and collect the cells by pipetting into a sterile tube.</li>
	<li>Count the number of isolated macrophages using a hemocytometer. Expect roughly 10 million macrophages per 10 cm plate. Calculate the total number of macrophages needed; each well on the 96-well shuttle requires 200,000 cells. Remember to add a few extra wells&#39; worth of cells to allow for pipetting losses.</li>
	<li>Pipette the calculated number of cells into a centrifuge tube. Centrifuge the cells for 10 min at 150 x g at room temperature. Do not centrifuge too quickly as this will diminish macrophage health and transfection efficiency.</li>
</ol>
5. Nucleofection&#160;of Macrophages with siRNA
<ol>
	<li>Using a sterile 96-well round bottom plate (which facilitates mixing while minimizing reagent loss), add 2 &#181;l of each siRNA to each well to be transfected. 
	Note: Depending on the number of siRNAs, this can be accomplished during the macrophage centrifugation step.</li>
	<li>Aspirate the media from the centrifuged cells. Gently resuspend the cells in Nucleofector&#8482; solution SF (containing the supplement solution), using 20 &#181;l per 200,000 cells pelleted. Once resuspended, use the cells immediately for optimal transfection.</li>
	<li>Transfer resuspended cells into a sterile trough; using a multichannel pipette, transfer 20 &#181;l of the resuspended cell mixture into each well of the 96-well plate containing the siRNAs. Gently mix by pipetting up and down.</li>
	<li>Transfer 20 &#181;l of the cell/siRNA mixture into the 96-well Nucleocuvette&#8482; plate. Critical step: avoid generating bubbles during the transfer step as this can induce errors during Nucleofection.</li>
	<li>After placing all of the experimental samples in the transfection plate, cover the 96-well plate with the provided lid and very gently tap on a hard surface to remove any bubbles from the samples.</li>
	<li>Place 96-well plate with lid into the 96-well Nucleofector shuttle and select &#39;upload and start&#39; on the bottom right of the program screen. Prior to Nucleofection, follow the program prompt to save the results. 
	Note: During the Nucleofection process, wells successfully tranfected will be depicted on the 96-well schematic on the screen by a green circle with a plus sign. A red circle with minus signs indicates an error, most likely due to bubbles or mis-pipetting the correct volume of reagents.</li>
</ol>
6. Recovery and Plating of Macrophages
<ol>
	<li>Immediately after Nucleofection, using a multichannel pipette, add 80 &#181;l of pre-warmed (37 &#186;C) RMP1-1640 media to each well. Add the media to the side of each well, allowing the media to slowly mix with the transfected cells, which are very delicate at this stage. Do not add the media too fast, as this will greatly decrease viability.</li>
	<li>Place the 96-well plates with transfected samples and RPMI-1640 into a 37 &#186;C incubator with 5% CO2 for 2 min; this is critical to allow the cells to recover prior to further manipulation.</li>
	<li>Remove the 96-well plate from the incubator and carefully transfer the mixture from each well into a 96-well tissue culture plate using a multichannel pipette.</li>
	<li>Using a multichannel pipette, add 100 &#181;l of pre-warmed (37 &#186;C) DMEM+FBS medium to each well.</li>
	<li>Incubate the 96-well tissue culture plate with samples in a 37 &#186;C incubator with 5% CO2 for 24-36 hr (optimize the exact timing for individual siRNAs, although this time range generally works well).</li>
</ol>
7. Stimulation of Macrophages with LPS
<ol>
	<li>Following the 24-36 hr post-Nucleofection&#160;incubation, prepare LPS (or other PAMPs) in fresh media. Prepare enough fresh medium for all the wells (200 &#181;l media/well of the 96-well plate plus a little extra for pipetting). Dilute LPS to a final concentration of 20 ng/ml in DMEM+FBS.</li>
	<li>Carefully aspirate media from the 96-well plate and add the fresh media containing the LPS to the cells.</li>
	<li>Monitor the response to LPS or other PAMPs at various times after stimulation. 6 hr is sufficient to generate a robust inflammatory cytokine response for cytokines such as IL-6 or TNF&#945;.</li>
</ol>
8. Monitoring the Induced Innate Immune Response, Gene Knockdown, and Viability in Macrophages
<ol>
	<li>To monitor LPS-induced cytokine production, pipette the supernatant from the cells into a 96-well storage plate. Cytokine production can then be measured by ELISA.</li>
	<li>Once the supernatant is removed, monitor cell viability using fluorescein diacetate (when cleaved by cellular esterases in live cells, this compound becomes fluorescent). Identify siRNAs that target genes that control cell viability using this approach. Note: the transfection process itself should have little effect on viability if performed properly.
	<ol>
		<li>Add fluorescein diacetate (5 mg/ml stock in acetone) to each well (1:100 final dilution in PBS).</li>
		<li>Incubate at room temperature for one to five min and then monitor fluorescence in the cells using a plate reader (460 nm excitation, 515 nm emission).</li>
		<li>Optional: To ensure that the fluorescence in the wells is uniform, lyse the cells using buffers such as RIPA; this provides a uniform fluorescent signal in the well, as some plate readers are configured to monitor only a small portion of the well; if the cells are not plated uniformly, the fluorescein diacetate could give an inaccurate reading.</li>
	</ol>
	</li>
	<li>As an alternative to monitoring cell viability, monitor siRNA-induced gene knockdown by qPCR using RNA derived from the adherent cells.
	<ol>
		<li>After the supernatant is removed from the cells (step 8.1 above), add RLT buffer (which is used for lysis and preservation of RNA) directly to the adherent cells to lyse them.</li>
		<li>Isolate RNA using a RNA preparation kit such as the RNeasy mini-kit, and use qPCR with gene-specific primers to monitor gene knockdown relative to control siRNA treatment. Use primers to &#914;actin or GAPDH for normalization.</li>
	</ol>
	</li>
	<li>As another alternative to monitor other aspects of the innate immune response, monitor the phagocytic ability of the macrophages by adding FITC-labeled E. coli particles directly onto the adherent cells using a kit. Following the manufacturer&#39;s protocol, which takes only a few hours, monitor phagocytic uptake using a plate reader or microscopy.</li>
</ol>

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The authors declare that they have no competing financial interests. This article was sponsored by Lonza, Inc.

Numerous studies have been published in which individual genes have been targeted by siRNA in murine macrophages. While lipid-mediated transfection has been used to deliver siRNA to macrophage cell lines on an individual basis, these methods suffer from potential effects on viability, limited gene knockdown, and variability from gene to gene. To develop more robust assays that could be used to target genes in medium- or high-throughput fashion, we optimized techniques using the Amaxa nucleofector 96-well shuttle system, ...

In this protocol, we describe efficient methods to inhibit genes in mouse macrophage cell lines using siRNAs and monitor the effects of these treatments on the innate immune response. These procedures are performed in 96-well format, allowing for RNAi screens in medium- or high-throughput fashion.
In response to infection, humans mount an immediate innate immune response and a slower but more specific adaptive immune response. This rapid innate immune response involves the recruitment and activation of phagocytic innate immune cells including macrophages1. Classically activated macrophages are involved in acute inflammatory responses and produce antimicrobial proteins and compounds, cytokines and chemokines, and present antigens2,3. Alternatively activated macrophages play a role in regulating immunity, maintaining tolerance, tissue repair, and wound healing4-8. Because of their wide array of functions, macrophages can play a role in numerous diseases including atherosclerosis, arthritis, and cancer9. Thus, the study of macrophages has been a key area of research for some time in a wide variety of disease fields.
Despite their importance in the innate immune response, macrophages can be challenging cells to work with. In particular, it is difficult to obtain efficient transfection using lipid reagents in macrophages without associated toxicity10,11. Moreover, even when siRNA is efficiently delivered to macrophages, the robustness of RNAi-induced gene knockdown often can be fairly moderate and can vary from gene to gene.
To overcome these technical challenges, we have optimized transfection and knockdown techniques12-16 in two mouse macrophage cell lines, RAW264.717 and J774A.118. This approach uses the Amaxa Nucleofector 96-well Shuttle System for transfection; this system uses a combination of specialized reagents and electroporation to transfect cells in 96-well format19. Following transfection, we describe methods to maximize cell recovery and viability and to maximize subsequent siRNA-induced gene knockdown. To illustrate the utility of this approach, we describe a protocol for siRNA delivery to these macrophage cell lines, stimulate these cells with lipopolysaccharide (LPS), and monitor the innate immune response at the level of production of several pro-inflammatory cytokines. We provide sample data in which we target the Toll-like receptor (TLR) family, whose members sense LPS and other pathogen associated molecular patterns (PAMPs), to regulate innate immunity.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

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.

To demonstrate the efficiency of transfection using this approach, we monitored uptake of FITC-labeled siRNA using a flow cytometer (Figure 1).
To illustrate the utility of our approach for monitoring the innate immune response, we transfected siRNAs targeting known innate immune regulatory genes into the RAW264.7 macrophage cell line, stimulated the cells with LPS, and then monitored production of the pro-inflammatory cytokines IL-6 and TNF&#945;. Different TLRs recognize dif...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

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

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 ...

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Immunology and Infection

10.6K Views.  National Jewish Health and University of Colorado School of Medicine. 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.

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

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

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 ...

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.

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). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...