We are grateful to Dr. Robert Heinzen for providing stocks of mCherry-expressing Coxiella burnetii. We thank Dr. Luis Teixeira for providing Invertebrate iridescent virus 6 and the Bloomington Stock Center for providing fly stocks. This project was funded in part by NIH grant R00 AI106963 (to A.G.G.) and Washington State University.

1.&#160;Ex vivo infection
<ol>
	<li>Medium and equipment
	<ol>
		<li>Under sterile conditions, prepare fresh Drosophila Hemocyte Isolating Medium (DHIM) containing 75% Schneider&#39;s Drosophila medium with 25% Fetal Bovine Serum (FBS) and filter sterilize it.</li>
		<li>Layer 2-3 pieces of 10 cm x 10 cm paraffin film under a stereomicroscope.</li>
		<li>Prepare the glass capillary. Set the capillary puller heater to 55% of maximum. Pull the capillary tube to a sharp point of approximately 10 &#181;m.</li>
		<li>Backfill the capillary with mineral oil.</li>
		<li>Assemble filled capillary tube onto the nanoinjector (Figure 1A), and open fused capillary tube tip by breaking off the tip with forceps (Figure 1B). The outer diameter of the tip should be 100 &#181;m for easy uptake of the hemocytes.</li>
		<li>Eject as much oil as possible from the capillary tube tip, then fill with DHIM. For easy visualization of the border of up-taken hemolymph and the oil, include an air bubble between the oil and DHIM (Figure 1B&#39;).</li>
	</ol>
	</li>
	<li>Hemolymph extraction
	<ol>
		<li>Pick 3rd instar Drosophila larvae from the inside wall of food vials gently using forceps and place them into a 100 &#181;m strainer (Figure 1C). 3rd instar larvae are found 3-6 days following fertile egg laying by an adult female. 
		NOTE: These experiments used the genotype, w1118;P{w+mC=Hml-GAL4.&#916;}2,P{w+mC=UAS-2xEGFP}AH2, since hemocytes from these animals express enhanced green fluorescent protein (EGFP) to aid in identification of the cells by microscopy.</li>
		<li>Pour 5 mL of sterile water over larvae and shake the strainer for 5 s. Place the strainer onto task wipe to remove the excess water (Figure 1C&#39;).</li>
		<li>Transfer the larvae into a 1.5 mL microcentrifuge tube. Anesthetize them with CO2 gas for 5 s (Figure 1D).</li>
		<li>Place the larvae onto paraffin film under the stereomicroscope, with dorsal-side facing up (Figure 2A).</li>
		<li>Place the glass capillary lightly onto the larval posterior body to hold it in place and disrupt the posterior cuticle open using fine pointed forceps (Figure 2B).</li>
		<li>Allow the hemolymph to flow onto the paraffin film (Figure 2C).</li>
		<li>Make a pool of hemolymph including hemocytes from 20-50 larvae at a time on the paraffin film.</li>
		<li>Take up pooled hemolymph using the glass capillary on the nanoinjector (Figure 2D). 
		NOTE: There should be approximately 10-20 &#181;L of hemolymph.</li>
		<li>Eject the hemolymph into a 1.5 mL microcentrifuge tube containing 500 &#181;L of DHIM (Figure 2E).</li>
		<li>Repeat steps 1.2.4) - 1.2.9) for every batch of larvae.</li>
	</ol>
	</li>
	<li>Count the number of hemocytes.
	<ol>
		<li>Pipette 5 &#181;L of 0.4 % Trypan blue solution in a 0.6 mL microcentrifuge tube to stain the dead cells. Gently mix the DHIM and hemocytes in the 1.5 mL tube using a pipette, and transfer 5 &#181;L of DHIM including hemocytes to the 0.6 mL microcentrifuge tube and mix gently.</li>
		<li>Pipette 10 &#181;L of cells from the 1:1 Trypan blue:hemocyte mixture into the hemocytometer.</li>
		<li>Count the number of live hemocytes that are not stained with Trypan blue in each of the 4 corner fields of the hemocytomter and calculate the concentration of the hemocytes per milliliter using formula: 
		<img alt="Equation 1" src="/files/ftp_upload/57077/57077eq1.jpg" /> 
		where X is the concentration of live hemocytes per milliliter; a, b, c, and d are the number of live cells (as determined by Trypan blue exclusion) in each 4 of the fields counted in the hemocytometer. Division by 2 of the total number of cells counted is due to the 1:1 dilution of the cells with Trypan blue. Cells stained with Trypan blue are considered dead.</li>
	</ol>
	</li>
	<li>Ex vivo Infections
	<ol>
		<li>Determine the number of wells to be seeded with cells based on timepoints and biological replicates needed for each experiment. 5.0&#215;104 hemocytes are desirable for RNA and protein purification following infection.</li>
		<li>Calculate the volume of pathogen stock to be diluted with DHIM for infection using the following formula: 
		<img alt="Equation 2" src="/files/ftp_upload/57077/57077eq2.jpg" /> 
		where Multiplicity of infection (MOI) is the number of viral or bacteria desired per cell. 
		NOTE: MOI used depends on the individual experiment and assays performed. Here, 10 genome equivalents (GE)/cell of C. burnetii, 10 CFU/cell of Listeria, or 1 TCID50/cell of IIV6 was used.</li>
		<li>Prepare 500 &#181;L of pathogen medium for each well of a 24-well plate by adding the proper volume of DHIM to the viral or bacterial volume in a tube.</li>
		<li>Place a 12 mm round cover glass (no. 1 thickness) in a well of a 24-well plate.</li>
		<li>Split the DHIM including the hemocytes into wells of a 24-well plate.</li>
		<li>Add 500 &#181;L of pathogen medium to hemocytes in a well.</li>
		<li>Centrifuge the plate at 1,000 x g for 5 min.</li>
		<li>Incubate the plate for 1 h at 28 &#176;C. Every 15 min, gently tilt the plate from back to front, then left to right for 5 s by hand.</li>
		<li>After the 1 h of invasion/attachment step 1.4.8), gently pipette off the pathogen medium and wash the hemocytes with fresh DHIM, and refill it with 500 &#181;L of fresh DHIM.</li>
		<li>Incubate the infected hemocytes for the desired time. In these experiments, C. burnetii-or IIV6-infected hemocytes are incubated for 24 h, and Listeria-infected hemocytes are incubated for 1, 2, or 4 h.</li>
	</ol>
	</li>
</ol>
2.&#160;In vivo infection
<ol>
	<li>Infection
	<ol>
		<li>Warm the Drosophila fruit juice agar plate at room temperature for 15 min. Plates are made as previously described20.
		<ol>
			<li>Add 30 g of agar to 700 mL of water and autoclave it for 40 min.</li>
			<li>Dissolve 0.5 g of methyl paraben in 10 mL of absolute ethanol.</li>
			<li>Add the methyl paraben solution to 300 mL of fruit juice concentrate.</li>
			<li>Quickly mix the juice concentrate into the autoclaved agar solution and dispense 5 mL into 10 &#215; 35 mm Petri dishes.</li>
			<li>After the plates have cooled for 15 min, store them at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Prepare 3rd instar larvae following steps 1.2.1) - 1.2.3).</li>
		<li>Place the yeast paste on an agar plate. Make a fine cut in the agar plate where larvae can migrate to avoid drying (Figure 3A). Assemble a 0.001 mm pointed tungsten needle with holding forceps using paraffin film (Figure 3B, C).</li>
		<li>Pipette 50 &#181;L of high-titer mCherry expressing-C. burnetii (5.95&#215;109&#160;GE/mL) onto the paraffin film under the stereo microscope and place the larvae into the pool of bacteria.</li>
		<li>Place the larvae into the pool of bacteria. Prick the larvae with a tungsten needle (Figure 3D). Transfer the larvae onto an agar plate (Figure 3E).</li>
		<li>Transfer the remaining pathogen medium onto the agar plate and seal the plate with paraffin film.</li>
		<li>Keep the larvae on the plate in moist air until the desired time post-infection (Figure 3F). In this experiment, C. burnetii-infected larvae are on the plate for 24 h.</li>
	</ol>
	</li>
	<li>Hemolymph extraction and plating of hemocytes
	<ol>
		<li>Prepare the medium and equipment following step 1.1).</li>
		<li>Place a 12 mm round cover glass (no. 1 thickness) in a well of a 24-well plate. Pipette 500 &#181;L of DHIM into the well.</li>
		<li>Extract the hemolymph from the infected larvae following steps 1.2.4) - 1.2.8).</li>
		<li>Eject the hemolymph into the well following step 2.2.2).</li>
		<li>Repeat 2.2.3) and 2.2.4) for multiple batches of larvae.</li>
		<li>Centrifuge the plate at 1,000 x g for 5 min.</li>
	</ol>
	</li>
</ol>
3. Visualization
<ol>
	<li>Fixing and staining
	<ol>
		<li>After allowing the hemocytes to settle on the round cover glass in the well, gently remove the medium from each well.</li>
		<li>Gently add 200 &#181;L of 4% paraformaldehyde (PFA) to each well of settled hemocytes. Incubate the hemocytes for 20 min at room temperature.</li>
		<li>Remove the 4% PFA and gently add 200 &#181;L of PBS containing 0.1% Triton X-100 and 1% Bovine Serum Albumin (BSA) to each well. Incubate the hemocytes for 10 min at room temperature.</li>
		<li>Remove the PBS and gently add 200 &#181;L of 1&#215; 4&#39;,6-diamidino-2-phenylindole (DAPI) to each well. Incubate the hemocytes for 10 min in the dark at room temperature.</li>
		<li>Remove the DAPI solution and gently add PBS to each well. Incubate the hemocytes for 5 min at room temperature.</li>
		<li>Drop 10 &#181;L of the antifade mounting medium on a glass microscope slide.</li>
		<li>After removing the PBS from each well, remove the cover glass from the 24-well plate using fine-pointed forceps. Gently place the cover glass onto the antifade mounting medium on the glass slide, with the hemocytes facing down.</li>
		<li>Allow the slide to dry by placing it in the dark overnight.</li>
	</ol>
	</li>
	<li>Confocal Imaging
	<ol>
		<li>Configure the confocal microscope for three color imaging of DAPI, EGFP, and mCherry. Use the following setting: DAPI excitation (ex) 405 nm, emission (em) 415-480 nm; EGFP ex 488 nm, em 493-564 nm; mCherry ex 587 nm, em 597-700 nm.</li>
		<li>Place the sample on the microscope and focus on the sample using a 63X/1.4 numerical aperture (NA) objective. Locate desired hemocytes in the field of view for imaging.</li>
		<li>Adjust laser power and detector gains to achieve appropriate exposure of the sample. Check multiple z-planes to ensure the exposure level is appropriate for the entire sample thickness.</li>
		<li>Find the top and bottom position on the z-axis of a whole hemocyte. Set these positions as the start and end positions for z-sectioning.</li>
		<li>Only use scanning zoom to image the area containing the hemocyte. Zoom factors of 3X are often used.</li>
		<li>Collect the image series at appropriate resolution such as 1024 x 1024 pixels in the x-y plane, and 0.3 &#181;m spacing in the z dimension.</li>
	</ol>
	</li>
	<li>3D model reconstruction 
	&#8203;NOTE: Open source software exists that performs many of the functions described below for 3D model reconstruction.
	<ol>
		<li>Import the z-sectioned image series file into the software associated with the confocal microscope for 3D model reconstruction.</li>
		<li>Select a cell showing co-localization of nuclei stained with DAPI and mCherry expressing C. burnetii in an EGFP expressing hemocyte. Crop the image series to contain only the single cell.</li>
		<li>Select the 3D Viewer option which reconstructs the 3D model using the software&#39;s pre-packaged algorithm. Choose the desired type of 3D representation among Blend, Surface, and Mixed options. In this method, hemocytes, nuclei, and C. burnetii are shown using surface models.</li>
		<li>Observe the 3D reconstructed cell from various viewing positions by holding the mouse button and dragging the cursor around the screen. Adjust the cell orientation and position of the modeled light source to optimize the image. Other options for Opacity, Minimum and Maximum Threshold, Specular, Ambient, Shineness, and Gamma exist to optimize the image.</li>
		<li>Take cross-sections through the model using clipping and sectioning commands to visualize the interior contents of the hemocyte.</li>
	</ol>
	</li>
</ol>
4. Application for gene and/or protein analysis
<ol>
	<li>Following the infection with IIV6 and Listeria, lyse cells for subsequent qRT-PCR or Western blot analysis as previously described21 and following manufacturer&#39;s instructions. 
	NOTE: Refer the Table of Materials for the primers for qRT-PCR and the antibodies for Western blot.</li>
	<li>Analyze PCR products by agarose gel electrophoresis, as previously described22, to ensure the proper length of the amplified product.</li>
</ol>

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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=The+Coxiella+burnetii+parasitophorous+vacuole.">The Coxiella burnetii parasitophorous vacuole.</a> Adv Exp Med Biol. 984, 141-169 (2012).</li><li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+prophenoloxidases+respond+inconsistently+to+Cu2++and+have+different+activity+in+vitro.">Drosophila melanogaster prophenoloxidases respond inconsistently to Cu2+ and have different activity in vitro.</a> Dev Comp Immunol. 36 (3), 619-628 (2012).</li><li>De Gregorio, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immune-responsive+Serpin+regulates+the+melanization+cascade+in+Drosophila.">An immune-responsive Serpin regulates the melanization cascade in Drosophila.</a> Dev Cell. 3 (4), 581-592 (2002).</li><li>Kari, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+raspberry+Gene+Is+Involved+in+the+Regulation+of+the+Cellular+Immune+Response+in+Drosophila+melanogaster.">The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster.</a> PLoS One. 11 (3), 0150910 (2016).</li><li>Wu, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+single-cell+RNA-sequencing+methods.">Quantitative assessment of single-cell RNA-sequencing methods.</a> Nat Methods. 11 (1), 41-46 (2014).</li><li>Buettner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+analysis+of+cell-to-cell+heterogeneity+in+single-cell+RNA-sequencing+data+reveals+hidden+subpopulations+of+cells.">Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells.</a> Nat Biotechnol. 33 (2), 155-160 (2015).</li><li>Nevil, M., Bondra, E. R., Schulz, K. N., Kaplan, T., Harrison, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+Binding+of+the+Conserved+Transcription+Factor+Grainy+Head+to+its+Target+Genes+Throughout+Drosophila+melanogaster+Development.">Stable Binding of the Conserved Transcription Factor Grainy Head to its Target Genes Throughout Drosophila melanogaster Development.</a> Genetics. 205 (2), 605-620 (2017).</li><li>Yang, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomes+of+lineage-specific+Drosophila+neuroblasts+profiled+by+genetic+targeting+and+robotic+sorting.">Transcriptomes of lineage-specific Drosophila neuroblasts profiled by genetic targeting and robotic sorting.</a> Development. 143 (3), 411-421 (2016).</li><li>Jaitin, D. 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The authors declare that they have no competing financial interests.

To better understand how host cells become infected, it is important to clarify the localization of pathogen in the cells, especially when experimenting on previously untested pathogen and cell type combinations4. While studying the cellular response cascade following infection can indicate productive pathogen invasion, the combination of imaging and cellular response data is essential to demonstrate pathogen invasion and infection. While reports showing 2D images of pathogen invasion into the hos...

Drosophila melanogaster is a well-established model organism for the study of innate immunity1. During the innate immune response, hemocytes play an important role in the response to pathogen challenge. Hemocytes are critical for encapsulating parasites, as well as having an important function in combating the pathogen through phagocytic action during fungal, viral, and&#160;bacterial infection2,3.
In order to best understand the host&#39;s innate immune response to pathogenic microbial infection, it is important to visualize how the pathogen invades host cells during infection. This visualization contributes to an understanding of the mechanism of invasion. Together with details of pathogen intracellular localization and the cellular response, these data can provide clues about the host response to infection and the cellular organelles with which the microbe interacts. Thus, 3D model reconstruction after imaging by microscopy can be helpful to determine the precise location of pathogens in host cells. In this study, we visualized the invasion of Coxiella burnetii (C. burnetii), the causative agent of Q fever, a zoonotic disease that poses a serious threat to both human and animal health, into primary Drosophila hemocytes. Recently, it was demonstrated that Drosophila are susceptible to the Biosafety level 2 Nine Mile phase II (NMII) clone 4 strain of C. burnetii and that this strain is able to replicate in Drosophila4, indicating that Drosophila can be used as a model organism to study C. burnetii pathogenesis.
Previous studies have used hemocytes to examine the host&#39;s innate immune response. Hemocytes have been used for morphological observations5,6,7, morphometric analysis2,8, phagocytosis analysis2,3, qRT-PCR2,9, immunoprecipitation10,11, immunofluorescent analysis10,12, immunostaining13, immunoblotting3,10,11 and immunohistochemistry9,14. Although Drosophila S2 cells are also available for various in vitro experiments, immortalization and potential pre-existing viral infection change their behavior15,16. The use of primary cells as opposed to an immortalized cell line, such as S2 cells, allows for the study of innate immune function in a system more representative of the whole organism. Additionally, the infection of hemocytes in vivo, prior to extraction, allows the cells to interact with other host proteins and tissue, an advantage over extraction of hemocytes prior to ex vivo infection. A number of different methods have been utilized to obtain a sufficient number of hemocytes in a short period of time to keep the hemocytes alive8,17,18,19.
In this study, we present a method to extract hemocytes from Drosophila 3rd instar larvae for pathogenic microbial infection with C. burnetii, Listeria monocytogenes (Listeria), or Invertebrate iridescent virus 6 (IIV6). We describe the methods for both in vivo and ex vivo hemocyte infections. In vivo- and ex vivo-infected hemocytes were visualized with confocal microscopy and used to build 3D models of C. burnetii invasion. Additionally, using the extraction protocol, ex vivo-infected hemocytes were used for gene and protein expression assays. Specifically, to examine the extent of infection with IIV6 and Listeria, total RNA or protein was isolated from the cells for qRT-PCR or Western blot analysis. Taken together, the protocol provides methods to rapidly collect high numbers of hemocytes from 3rd instar larvae and evidence that primary hemocytes, infected either in vivo or ex vivo, are a suitable platform for microbial pathogen infection studies and applicable downstream analyses such as microscopy, transcriptomics, and proteomics.

<table border="0" cellpadding="0" cellspacing="0" style="width:1128px;" width="1125">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:303px;">Schneider&#39;s Drosophila Medium&#160;</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">21720024</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:288px;">GE Healthcare Life Sciences&#160; (HyClone)</td>
			<td style="width:145px;">SH30070.03HI</td>
			<td style="width:179px;">1.1.1), 2.1.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Filter (0.22 &#181;L)</td>
			<td style="width:288px;">RESTEK</td>
			<td style="width:145px;">26158</td>
			<td style="width:179px;">1.1.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Strainer (100 &#181;m)</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">542000</td>
			<td>1.2.1), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Stereo microscope</td>
			<td style="width:288px;">Amscope</td>
			<td style="width:145px;">SM-1BSZ-L6W</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass capillary</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">21-171-4</td>
			<td style="width:179px;">1.1), 1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Capillary puller</td>
			<td style="width:288px;">Narishige International USA, Inc.</td>
			<td style="width:145px;">PC-10</td>
			<td style="width:179px;">1.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mineral oil</td>
			<td style="width:288px;">Snow River Products</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Nanoinjector</td>
			<td style="width:288px;">Drummond Scientific Company</td>
			<td style="width:145px;">3-000-204</td>
			<td style="width:179px;">1.1), 1.2), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">82027-402</td>
			<td style="width:179px;">1.1.5), 1.2), 2), 3.1.7)</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:303px;">CO2 delivery apparatus</td>
			<td style="width:288px;">Genesee Scientific</td>
			<td style="width:145px;">59-122BC</td>
			<td style="width:179px;">1.2), 2)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Trypan Blue</td>
			<td style="width:288px;">Thermo Fisher Scientific (Gibco)</td>
			<td style="width:145px;">15250061</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemocytometer</td>
			<td style="width:288px;">Hausser Scientific</td>
			<td style="width:145px;">3100</td>
			<td style="width:179px;">1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">24 well plate</td>
			<td style="width:288px;">Greiner bio-one</td>
			<td style="width:145px;">662160</td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coxiella burnetii - mCherry</td>
			<td style="width:288px;">Dr. Heinzen, R.</td>
			<td style="width:145px;"></td>
			<td style="width:179px;">1.4), 2.2)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:303px;">Drosophila fruit juice plates</td>
			<td style="width:288px;">Cold Spring Harbor Protocols</td>
			<td style="width:145px;"></td>
			<td>2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Agar</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP1423-500</td>
			<td style="width:179px;">2.1.1.1)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Methyl paraben</td>
			<td style="width:288px;">Amresco</td>
			<td style="width:145px;">0572-500G</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Absolute ethanol</td>
			<td style="width:288px;">Fisher Bioreagents</td>
			<td style="width:145px;">BP2818-500</td>
			<td style="width:179px;">2.1.1.2)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Welch&#39;s 100% Grape juice frozen concentrate, 340 mL</td>
			<td style="width:288px;">Amazon</td>
			<td style="width:145px;">B0025UJVGM</td>
			<td style="width:179px;">2.1.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Petri dishes, 10 x 35 mm</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">08-757-100A</td>
			<td style="width:179px;">2.1.1.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope cover glass</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-545-80</td>
			<td style="width:179px;">1.4.4), 2.2.2)</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:303px;">Yeast, Bakers Dried Active</td>
			<td style="width:288px;">MP Biomedicals</td>
			<td style="width:145px;">0210140001</td>
			<td>2.1) Add 2 parts of water to 1 part of yeast (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tungsten needle</td>
			<td style="width:288px;">Fine Science Tools</td>
			<td style="width:145px;">10130-20</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Holding forceps</td>
			<td style="width:288px;">VWR</td>
			<td style="width:145px;">HS8313</td>
			<td style="width:179px;">2.1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">FLO4042-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP151-500</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">BP9706-100</td>
			<td style="width:179px;">3.1.3)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">62247</td>
			<td style="width:179px;">3.1.4)</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:303px;">Antifade mounting medium</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">P36930</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;width:303px;">Confocal microsope</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">TCS SP8-X White Light Confocal Laser Scanning Microscope</td>
			<td style="width:179px;">3.2)</td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:303px;">3D imaging reconstruction software</td>
			<td style="width:288px;">Leica</td>
			<td style="width:145px;">LASX with 3D visualization module</td>
			<td style="width:179px;">3.3)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope slides</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:145px;">12-552-3</td>
			<td style="width:179px;">3.1.6)</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:303px;">Invertebrate iridescent virus 6 (IIV6)</td>
			<td style="width:288px;">Dr. Teixeria, L.</td>
			<td style="width:145px;"></td>
			<td>4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008)</td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:303px;">Listeria monocytogenes</td>
			<td style="width:288px;">ATCC</td>
			<td style="width:145px;">strain: 10403S</td>
			<td>4) Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 &#181;g/ml streptomycin at 30 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">DNase I</td>
			<td style="width:288px;">Thermo Fisher Scientific(Invitrogen)</td>
			<td style="width:145px;">18068015</td>
			<td>gDNA degradation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">cDNA Synthesis Kit</td>
			<td style="width:288px;">Bio-Rad</td>
			<td style="width:145px;">1708891</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_F</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TCT TGT TTT CAG AAC CCC ATT -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">IIV6_193R_R</td>
			<td style="width:288px;">IDT</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- CAC GAA GAA TGA CCA CAA GG -3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_fwd</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- GAA GCG TTT CTC CAA ACG -AG</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">RpII_qRTPCR_rev</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;"></td>
			<td>qRT-PCR, 5&#39;- TTG AGC GTA AGC ATC ACC -TG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SYBR Green qRT-PCR reagent</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">K0251, K0252, K0253</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Real-Time PCR System</td>
			<td style="width:288px;">Thermo Fisher Scientific</td>
			<td style="width:145px;">4351107, 7500 Software v2.0</td>
			<td>qRT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Listeria monocytogenes antibody</td>
			<td style="width:288px;">abcam</td>
			<td style="width:145px;">ab35132</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Actin antibody produced in rabbit</td>
			<td style="width:288px;">SIGMA-ALDRICH</td>
			<td style="width:145px;">A2066</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Anti-Rabbit IgG (H+L), HRP Conjugate</td>
			<td style="width:288px;">Promega</td>
			<td style="width:145px;">W4011</td>
			<td>Western blot</td>
		</tr>
	</tbody>
</table>

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.

To collect live hemocytes for ex vivo infection, up to 3&#215;106 hemocytes were extracted from 200 Drosophila 3rd instar larvae. To develop our method, a number of different techniques were attempted. Individual larval dissection would take up to 1.5 h, and an average of ~8000 cells were obtained using this method18, most of which were not alive by the end of collection. Next, we tried to extract hemolymph, which contained t...

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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.

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 ...

extraction-hemocytes-from-drosophila-melanogaster-larvae-for

Research

JoVE Journal

Immunology and Infection

11.6K Views.  Washington State University. 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.

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

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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 ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

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 ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

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 ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

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

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A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

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

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice