We thank members of the Department of Biological Sciences at George Washington University (GWU) for critical reading of the manuscript. GT was supported through a Harlan summer fellowship from GWU. All graphical figures were made using BioRender.

1. Fly rearing
NOTE: The D. melanogaster life cycle is divided into four stages: embryo, larva, pupa, and adult. The generation time with optimal rearing conditions in the laboratory (~25 &#176;C, 60% humidity, and sufficient food) is approximately 10 days from fertilized egg to eclosed adult.Females lay ~100 embryos per day, and embryogenesis lasts about 24 h22. The larvae undergo three developmental stages (instars; L1-L3) in ~4 days (L1 and L2: 24 h, and L3: 48 h). The first instar larvae begin to feed immediately on the surface of the medium. Second instar larvae burrow into the medium, whereas third instar larvae leave the medium and wander up the vial walls, looking for a place to pupariate for 24-48 h. The D. melanogaster line used for this protocol is Oregon R (FBsn0000276).
<ol>
	<li>Add dry food containing a cornmeal-soy-based diet to a narrow polystyrene vial (25 mm x 95 mm) to aboue 1/5 to 2/5 volume. Then add 9 mL of water and allow the vial to sit for 1 min until the diet is completely hydrated.</li>
	<li>Add about 10 granules of dry baker&#39;s yeast to the vial and place a mixture of at least 20 newly emerged male and female adult flies.</li>
	<li>Incubate the vial at 25 &#176;C on a 12:12-h light: dark photoperiodic cycle.</li>
	<li>To ensure life cycle progression, check the fly vials daily and record the initial development stages.</li>
</ol>
2. Larvae selection for infection
<ol>
	<li>Select larvae with a fine paintbrush (camel&#39;s hair is best) once they reach the wandering third instar stage on the day the infection will be carried out (Figure 1).</li>
	<li>Wash the selected larvae with Ringer&#39;s solution (100 mM NaCl, 1.8 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES pH 6.9) in a small Petri dish (60 mm x 15 mm) upon removal from their original vial23.</li>
	<li>Place the larvae on filter paper (150 mm diameter) that is slightly moistened with around 5 mL of Ringer&#39;s solution in a Petri dish (100 mm x 15 mm) (Figure 2A).</li>
	<li>Add ~1 mL of Ringer&#39;s solution daily to the filter paper as needed to avoid dryness and larval desiccation.</li>
</ol>
3. Bacterial preparation
<ol>
	<li>Culture P. asymbiotica bacteria on Luria Bertani (LB) agar media at 28 &#176;C for 48 h.</li>
	<li>Use a biosafety level 2 cabinet to transfer a single colony of P. asymbiotica to inoculate a liquid culture in 10 mL of LB media.</li>
	<li>Incubate the P. asymbiotica liquid culture overnight at 28 &#176;C in a rotary shaker set to 220 rpm.</li>
	<li>Culture E. coli bacteria in a similar fashion, but carry out the initial growth on agar media and liquid culture incubation at 37 &#176;C overnight.</li>
	<li>Centrifuge each overnight bacterial culture for 3 min at 13,000 x g and 4 &#176;C.</li>
	<li>Discard the supernatant and wash the resulting bacterial pellets three times with 10 mL of sterile PBS.</li>
	<li>Centrifuge the pellets again for 3 min at 13,000 x g and 4 &#176;C.</li>
	<li>Dilute each pellet with sterile PBS to adjust bacterial concentrations to the optical density (600 nm) of 0.25 for P. asymbiotica and 0.015 for E. coli, which correspond to 100-300 colony forming units per larva of each bacterial preparation, using a spectrophotometer.</li>
</ol>
4. Injector preparation
<ol>
	<li>Prepare capillaries using 3.5&#8243; glass capillary tubes and a micropipette puller. Set the instrument as follows: heat: 580; pull: 143; velocity: 25; delay: 1; pressure: 500.</li>
	<li>Use straight serrated medium point forceps to open the glass capillary to the degree that will allow the delivery of the experimental treatments while causing minimal damage to the larvae (Figure 2B). To optimize this procedure while practicing injections, use Trypan Blue solution (dilute the stock 0.4% with PBS to 0.2%) to easily track leakage while injecting and the survival of the blue larvae.</li>
	<li>Fill the capillary with mineral oil using a plastic, disposable 20 mL syringe with a hypodermic needle (22 G, 25 mm length).</li>
	<li>Set up the nanoliter injector and eject the oil out of the capillary (Figure 2C).</li>
	<li>Fill the capillary with the desired bacterial preparation for injection. Take the solution from a drop (~20 &#181;L) that is placed on parafilm. In this protocol, 50.2 nL of two bacterial stocks were injected.</li>
</ol>
5. Larvae injection
<ol>
	<li>Anesthetize D. melanogaster larvae using carbon dioxide for ~2 min prior to the procedure.</li>
	<li>Gently transfer the anesthetized larvae to a filter paper moistened in Ringer&#39;s solution in preparation for injection under a stereomicroscope. The larvae will be lethargic or passive at this point.</li>
	<li>To inject larvae, apply firm pressure on the dorsal side of the posterior end (the tracheal spiracles are apparent at the posterior end versus black mouthparts at the anterior end) using forceps (Figure 2D).</li>
	<li>Insert the needle horizontally towards the posterior end of the larvae, near the cuticle. A successful injection will not result in a leakage of the injected solution from the larvae (Figure 3).</li>
	<li>Remove the forceps that were used to apply pressure on the larvae&#39;s tail before withdrawing the needle. If not removed first, the forceps can force hemolymph and/or the intestine out of the larvae from the wound site.</li>
	<li>Infect the appropriate number of larvae for the study being performed. In this particular protocol, 20 larvae for each experimental condition were injected.</li>
	<li>Using forceps, gently transfer the injected larvae to a separate moist filter paper in a Petri dish (10 larvae per dish) or a food vial (depending on the purpose of the experiment) to allow for recovery.</li>
	<li>Place the Petri dishes or food vials in an incubator at 25 &#176;C. If larvae are kept in a Petri dish, add Ringer&#39;s solution as necessary to prevent desiccation.</li>
</ol>
6. Recording survival/mortality
<ol>
	<li>Record the number of dead and live D. melanogaster larvae according to the set experimental time points. Live larvae will continue to develop into pupae and adults.</li>
	<li>Use statistical programs, such as Prism, to enter the raw survival/mortality data, analyze them with the log-rank (Mantel-Cox) test, and represent the results in figures.</li>
</ol>

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M., O'Halloran, D., Eleftherianos, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterorhabditis+bacteriophora+excreted-secreted+products+enable+infection+by+Photorhabdus+luminescens+through+suppression+of+the+Imd+pathway.">Heterorhabditis bacteriophora excreted-secreted products enable infection by Photorhabdus luminescens through suppression of the Imd pathway.</a> Frontiers in Immunology. 10, 2372 (2019).</li><li>Cherry, S., Silverman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+in+Drosophila:+New+tricks+from+an+old+friend.">Host-pathogen interactions in Drosophila: New tricks from an old friend.</a> Nature Immunology. 7 (9), 911-917 (2006).</li><li>Younes, S., Al-Sulaiti, A., Nasser, E., Najjar, H., Kamareddine, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+organism+in+host-pathogen+interaction+studies.">Drosophila as a model organism in host-pathogen interaction studies.</a> Frontiers in Cellular and Infection Microbiology. 10, 214 (2020).</li><li>Kenney, E., Hawdon, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasitoid+wasp+of+Drosophila+employs+preemptive+and+reactive+strategies+to+deplete+its+host's+blood+cells.">Parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host's blood cells.</a> PLoS Pathogens. 17 (5), 1009615 (2021).</li><li>Vlisidou, I., Wood, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+blood+cells+and+their+role+in+immune+responses.">Drosophila blood cells and their role in immune responses.</a> The FEBS Journal. 282 (8), 1368-1382 (2015).</li><li>Harnish, J. 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S., Br&#252;ckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+cellular+immunity+in+Drosophila+melanogaster.">Macrophages and cellular immunity in Drosophila melanogaster.</a> Seminars in Immunology. 27 (6), 357-368 (2015).</li><li>Dudzic, J. P., Kondo, S., Ueda, R., Bergman, C. M., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+innate+immunity:+regional+and+functional+specialization+of+prophenoloxidases.">Drosophila innate immunity: regional and functional specialization of prophenoloxidases.</a> BMC Biology. 13, 81 (2015).</li><li>Honti, V., Csord&#225;s, G., Kurucz, &. #. 2. 0. 1. ;., M&#225;rkus, R., And&#243;, 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+cell-mediated+immunity+of+Drosophilamelanogaster:+hemocyte+lineages,+immune+compartments,+microanatomy+and+regulation.">The cell-mediated immunity of Drosophilamelanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.</a> Developmental and Comparative Immunology. 42 (1), 47-56 (2014).</li><li>Siva-Jothy, J. A., Prakash, A., Vasanthakrishnan, R. B., Monteith, K. 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The authors declare no competing interests.

Drosophila melanogaster is among the most valuable, experimentally manipulated models used for investigations of innate immunity and pathogenesis of various microbial infections. This is due to its simple and fast life cycle, simple upkeep in a laboratory, well-established evolutionary genetics, and diverse genetic toolbox. Previous methods of D. melanogaster larvae injections, such as using a hybrid microfluidic device or a Narishige micromanipulator, require highly specialized equipment and can be cos...

Drosophila melanogaster has been immensely utilized in biological and biomedical research for several decades, as the sophisticated array of genetic and molecular tools have steadily evolved for analysis of a wide range of studies1,2,3,4. The evolutionarily conserved aspects of development, homeostasis and innate immunity in D. melanogaster have made it a valuable model organism for studying various human and insect diseases5,6. Notably, the fundamental role of the D. melanogaster model for studying immunity has been largely exemplified in adult flies studies. However, D. melanogaster larvae studies have also contributed to the current knowledge and mainly explored cellular immune responses, specifically for wasp and nematode infections that occur through the insect cuticle7,8,9,10. Drosophila melanogaster larvae possess three different types of blood cells, collectively called hemocytes: plasmatocytes, crystal cells, and lamellocytes11,12,13. These cells can mount an array of immune responses when D. melanogaster larvae are infected with pathogens such as bacteria, fungi, viruses, and parasites14,15,16. Cellular immune responses include direct engulfment (phagocytosis) of small molecules or bacteria, melanization, encapsulation of larger pathogens such as parasitoid eggs, and production of reactive oxygen species (ROS) and Nitric oxide synthases (NOS)17,18,19.
In contrast, fewer studies have been published on the use of the D. melanogaster larval model to analyze humoral immune responses. This is mainly due to the application of feeding assays for oral infection of D. melanogaster larvae and several challenges associated with microinjecting larvae including the precise handling of larvae and proper use of the microneedle, especially during penetration20,21. Thus, the limited knowledge of larval infection and technical difficulties (i.e., high mortality) have frequently made the D. melanogaster larval model difficult to use. A larval model will have the potential to identify novel molecular mechanisms that will provide further insights into host-pathogen interactions and the induction of specific host innate immune responses against pathogenic infections.
Here a simple and efficient protocol that can be used to inject D. melanogaster larvae with various pathogens, such as bacteria, is described in detail. In particular, D. melanogaster larvae are used for injections with the human pathogen Photorhabdus asymbiotica and the non-pathogenic bacteria Escherichia coli. This method can be used for the manipulation and analysis of D. melanogaster&#39;s immune responses to various microbial infections.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fly Food B (Bloomington Recipe)</td>
			<td>LabExpress</td>
			<td>7001-NV</td>
			<td>Food B, in narrow vials, 100 vials/tray</td>
		</tr>
		<tr>
			<td>100 x 15, Mono Petri Dishes Fully Stackable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td>Diameter 100 x 15 mm</td>
		</tr>
		<tr>
			<td>60 x 15, Mono Petri dishes Fully Stackable</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td>Diameter 60 x 15 mm</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>VWR</td>
			<td>53440-186</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 1 qualitative filter paper standard grade, circle</td>
			<td>VWR</td>
			<td>28450-150</td>
			<td>Diameter 150 mm</td>
		</tr>
		<tr>
			<td>Lab culture Class II Type A2 Biosafety Safety Cabinet</td>
			<td>ESCO</td>
			<td>LA2-4A2-E</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1425-500</td>
			<td>LB agar miller powder 500 g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher Scientific</td>
			<td>BP1426-500</td>
			<td>LB broth miller powder 500 g</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Alfa Aesar, Thermo Fisher Scientific</td>
			<td>31911-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond</td>
			<td>3-000-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow Drosophila Vials, Polystyrene</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles, hypodermic</td>
			<td>VWR</td>
			<td>89219-316</td>
			<td>22 G, 25 mm</td>
		</tr>
		<tr>
			<td>Next Generation Micropipette Puller</td>
			<td>World Precision Instruments</td>
			<td>SU-P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>VWR</td>
			<td>97062-732</td>
			<td>Buffer PBS tablets biotech grade 200tab</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>Version 8</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes - plastic, disposable</td>
			<td>VWR</td>
			<td>76124-652</td>
			<td>20 mL</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

drosophila melanogaster larva injection protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

When performed correctly, injections of D. melanogaster larvae show a bacterium-specific effect. The survival data were collected at several time points following infections of P. asymbiotica (strain ATCC43943), E. coli (strain K12), and PBS (Figure 4). Whereas D. melanogaster larvae are susceptible to P. asymbiotica, which compromises survival rapidly, larvae injected with E. coli or PBS controls exhibit prolonged survivals<sup class="xr...

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Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

drosophila-melanogaster-larva-injection-protocol

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JoVE Journal

Immunology and Infection

5.8K Views.  The George Washington University. Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described. 

Video: Drosophila melanogaster Larva Injection Protocol

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...