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 host cells tends to indicate productive infection, some questions may remain regarding the timing of initial pathogen invasion in host cells. Thus, 3D models reconstructed from z-section scanning of 2D images can address these questions and indicate pathogen location in the host cells (Figures 4 and 5). However, a limitation of using primary hemocytes is their longevity in cell culture. A previous report states that hemocyte survival in cell culture media is only 8 h18, and in our infection protocol, we were able to perform assays up to 24 h, but not longer. Therefore, it was not possible to observe high levels of C. burnetii replication or the formation of the parasitophorous vacuole which does not begin to form until 2 days post-infection and is not observably large until 4-6 days post-infection30.
For many experiments where the endpoint assays utilize protein or RNA for analysis, large numbers of cells are usually required to produce sufficient material for interrogation. For example, here, we use endpoint analyses such as Western blotting and qRT-PCR to probe the extent of pathogen infection in primary hemocytes derived from 3rd instar Drosophila larvae. Thus, the method presented here focuses on rapid larval dissection and the collection of the hemolymph containing sufficient live hemocytes for ex vivo pathogen infection. This method introduces the use of a nanoinjector to take up the hemolymph and hemocytes quickly. Placing the hemocytes in DHIM containing 25% FBS is important for hemocyte survival. Additionally, for the ex vivo infections presented here, a large number of hemocytes are needed. To avoid melanization31,32,33, dissection and hemocyte collection must occur rapidly. While other methods for hemocyte extraction exist18,19,23, the duration of these methods to collect a sufficiently large number of hemocytes for ex vivo infection was too long. The 100 &#181;m fine tip is beneficial for the uptake of hemolymph without taking up other organs that may lead to clogging of the capillary needle. The use of a nanoinjector may also be automated when it is attached to a micromanipulator stage and foot pedal, allowing the user to focus on quick dissection of the Drosophila larvae and decrease the time that the hemocytes are without surrounding larval tissue or cell culture media. Nevertheless, the use of a pipette is also available to take up the hemolymph for transfer to DHIM. In addition, our method utilizes CO2 gas to anesthetize larvae prior to dissection; the use of a cold block with paraffin film covering is another viable method23. Finally, dissection of larvae in a drop of DHIM during the release of the hemolymph may reduce the number of hemocytes that are lost from sticking to the paraffin film but will increase time needed for uptake by the capillary needle.
A common immune response in Drosophila to injury, which occurs during the opening and dissection of the larval cuticle, is melanization31,32,33. During the extraction of hemocytes, we would observe melanization of the hemocytes in the DHIM as early as 10 min following extraction. As melanization is a rapid process, these cells would be excluded from the pathogen infection experiments. Additionally, the anti-coagulant phenylthiourea can be added to DHIM to inhibit the phenoloxidase-activating system and melanization during wounding9. Nevertheless, as melanization and apoptosis are innate immune responses of Drosophila, their levels can be quantified following hemocyte extraction and stimulation using the methods described here.
While recovering large amounts of protein or RNA material is a requirement for techniques such as Western blot and qRT-PCR, new techniques, such as RNAseq require much less input material, as little as 10 pg of high-quality total RNA from a single cell34. Experiments such as these raise interesting experimental questions, and since Drosophila hemocytes are a heterogeneous population containing crystal cells, plasmatocytes, and lamellocytes7, one could begin to interrogate the transcriptional response of each cell type35. For example, Kurucz et al., have developed antibodies that could be used to isolate the Drosophila hemocyte subsets that could be used for transcriptional or proteomic profiling following various stimuli. Additionally, the recent development of single cell RNAseq technology could define transcriptomes of each cell type without the use of antibodies to initially separate each cell type36,37,38,39. Furthermore, one could ask questions regarding gene regulation in the hemocyte subsets that may help us understand how human blood cell lineages originated and evolved from ancient organisms through comparative genomics efforts. Tackling problems such as these requires the combination of a wide range of experimental techniques, and the technique described here may be useful for such efforts when the isolation of a large number of hemocytes from invertebrate larvae is required.
Here, we suggest the combination of 3D models with numerical, biochemical data for confirmation of infection. In future studies, we can use the methods described here to observe immune responses and the mechanism of pathogen invasion into host cells by co-staining host proteins for microscopy analysis.

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><tbody><tr><td>Schneider&#039;s &lt;em&gt;Drosophila&lt;/em&gt; Medium&amp;nbsp;</td><td>Thermo Fisher Scientific (Gibco)</td><td>21720024</td><td>1.1.1), 2.1.2)</td></tr><tr><td>Fetal Bovine Serum</td><td>GE Healthcare Life Sciences&amp;nbsp; (HyClone)</td><td>SH30070.03HI</td><td>1.1.1), 2.1.2)</td></tr><tr><td>Filter (0.22 &amp;micro;L)</td><td>RESTEK</td><td>26158</td><td>1.1.1)</td></tr><tr><td>Strainer (100 &amp;micro;m)</td><td>Greiner bio-one</td><td>542000</td><td>1.2.1), 2)</td></tr><tr><td>Stereo microscope</td><td>Amscope</td><td>SM-1BSZ-L6W</td><td>1.2), 2)</td></tr><tr><td>Glass capillary</td><td>Fisher Scientific</td><td>21-171-4</td><td>1.1), 1.2), 2)</td></tr><tr><td>Capillary puller</td><td>Narishige International USA, Inc.</td><td>PC-10</td><td>1.1.3)</td></tr><tr><td>Mineral oil</td><td>Snow River Products</td><td></td><td>1.1.4)</td></tr><tr><td>Nanoinjector</td><td>Drummond Scientific Company</td><td>3-000-204</td><td>1.1), 1.2), 2.2)</td></tr><tr><td>Forceps</td><td>VWR</td><td>82027-402</td><td>1.1.5), 1.2), 2), 3.1.7)</td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;delivery apparatus</td><td>Genesee Scientific</td><td>59-122BC</td><td>1.2), 2)</td></tr><tr><td>Trypan Blue</td><td>Thermo Fisher Scientific (Gibco)</td><td>15250061</td><td>1.3)</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>3100</td><td>1.3)</td></tr><tr><td>24 well plate</td><td>Greiner bio-one</td><td>662160</td><td>1.4), 2.2)</td></tr><tr><td>&lt;em&gt;Coxiella burnetii&lt;/em&gt; - mCherry</td><td>Dr. Heinzen, R.</td><td></td><td>1.4), 2.2)</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; fruit juice plates</td><td>Cold Spring Harbor Protocols</td><td></td><td>2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full</td></tr><tr><td>Agar</td><td>Fisher Bioreagents</td><td>BP1423-500</td><td>2.1.1.1)</td></tr><tr><td>Methyl paraben</td><td>Amresco</td><td>0572-500G</td><td>2.1.1.2)</td></tr><tr><td>Absolute ethanol</td><td>Fisher Bioreagents</td><td>BP2818-500</td><td>2.1.1.2)</td></tr><tr><td>Welch&#039;s 100% Grape juice frozen concentrate, 340 mL</td><td>Amazon</td><td>B0025UJVGM</td><td>2.1.1.3)</td></tr><tr><td>Petri dishes, 10 x 35 mm</td><td>Fisher Scientific</td><td>08-757-100A</td><td>2.1.1.4)</td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12-545-80</td><td>1.4.4), 2.2.2)</td></tr><tr><td>Yeast, Bakers Dried Active</td><td>MP Biomedicals</td><td>0210140001</td><td>2.1) Add 2 parts of water to 1 part of yeast (v/v)</td></tr><tr><td>Tungsten needle</td><td>Fine Science Tools</td><td>10130-20</td><td>2.1)</td></tr><tr><td>Holding forceps</td><td>VWR</td><td>HS8313</td><td>2.1)</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>FLO4042-500</td><td>3.1.3)</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td>3.1.3)</td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP9706-100</td><td>3.1.3)</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>Thermo Fisher Scientific</td><td>62247</td><td>3.1.4)</td></tr><tr><td>Antifade mounting medium</td><td>Thermo Fisher Scientific</td><td>P36930</td><td>3.1.6)</td></tr><tr><td>Confocal microsope</td><td>Leica</td><td>TCS SP8-X White Light Confocal Laser Scanning Microscope</td><td>3.2)</td></tr><tr><td>3D imaging reconstruction software</td><td>Leica</td><td>LASX with 3D visualization module</td><td>3.3)</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-552-3</td><td>3.1.6)</td></tr><tr><td>Invertebrate iridescent virus 6 (IIV6)</td><td>Dr. Teixeria, L.</td><td></td><td>4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008)</td></tr><tr><td>&lt;em&gt;Listeria monocytogenes&lt;/em&gt;</td><td>ATCC</td><td>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 &amp;micro;g/ml streptomycin at 30 &amp;deg;C.</td></tr><tr><td>DNase I</td><td>Thermo Fisher Scientific(Invitrogen)</td><td>18068015</td><td>gDNA degradation</td></tr><tr><td>cDNA Synthesis Kit</td><td>Bio-Rad</td><td>1708891</td><td>cDNA synthesis</td></tr><tr><td>IIV6_193R_F</td><td>IDT</td><td></td><td>qRT-PCR, 5&#039;- TCT TGT TTT CAG AAC CCC ATT -3&#039;</td></tr><tr><td>IIV6_193R_R</td><td>IDT</td><td></td><td>qRT-PCR, 5&#039;- CAC GAA GAA TGA CCA CAA GG -3&#039;</td></tr><tr><td>RpII_qRTPCR_fwd</td><td>SIGMA-ALDRICH</td><td></td><td>qRT-PCR, 5&#039;- GAA GCG TTT CTC CAA ACG -AG</td></tr><tr><td>RpII_qRTPCR_rev</td><td>SIGMA-ALDRICH</td><td></td><td>qRT-PCR, 5&#039;- TTG AGC GTA AGC ATC ACC -TG</td></tr><tr><td>SYBR Green qRT-PCR reagent</td><td>Thermo Fisher Scientific</td><td>K0251, K0252, K0253</td><td>qRT-PCR</td></tr><tr><td>Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>4351107, 7500 Software v2.0</td><td>qRT-PCR</td></tr><tr><td>Anti-Listeria monocytogenes antibody</td><td>abcam</td><td>ab35132</td><td>Western blot</td></tr><tr><td>Anti-Actin antibody produced in rabbit</td><td>SIGMA-ALDRICH</td><td>A2066</td><td>Western blot</td></tr><tr><td>Anti-Rabbit IgG (H+L), HRP Conjugate</td><td>Promega</td><td>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 the hemocytes, from 20 larvae at a time using a glass capillary tube19, but the capillary became clogged with cuticle material and the hemolymph was not able to be efficiently taken up by the glass capillary. Finally, we mechanically disrupted the cuticle of 20-50 larvae per batch with fine forceps12, and made a pool of hemolymph for easy uptake. This allowed easy collection of a large amount of hemocytes from a large number of larvae (Table 1).
To indicate that the extracted hemocytes were alive and suitable for the infection, a Trypan blue exclusion assay was performed to calculate the percentage of live hemocytes extracted using the method presented here (Figure 6A). In addition, we compared our method for hemocyte extraction with a previously published method where the goal was to develop a mechanical disruption method to isolate and differentiate between circulating and resident hemocytes from individual Drosophila larva23. Upon comparison between the two methods from 10 independent experimental isolations, we observed that cell viability was slightly greater using the method presented here (Figure 6A); however, the method from Petraki et al. yielded close to twice as many hemocytes from every 10 dissected larvae (Figure 6B). A reason for the increased number of hemocytes from the Petraki et al. method is that this method collects resident (sessile) hemocytes, as well as circulating hemocytes. To confirm that the cells extracted with the method presented here were in fact hemocytes, we utilized a fly line containing transgenes for the hemolectin (Hml) promoter driving GAL4 in circulating hemocytes that binds the upstream activator sequence (UAS) to activate enhanced green fluorescent protein (EGFP) transcription and expression in the hemocytes. Hemocytes from hml-GAL4&#62;UAS-EGFP larvae were extracted onto chambered coverglass slides, fixed, permeabilized and stained with DAPI as previously described21. Figure 7 shows that most DAPI-positive cells are also EGFP-positive. Quantification of co-expression was performed from multiple images, from which we calculated that 86&#177;9% of cells isolated were hemocytes.
The protocol in innovative 3D models of pathogen invasion into Drosophila hemocytes extracted from 3rd instar larvae following ex vivo or in vivo C. burnetii infection. We were able to image C. burnetii infection since the bacteria express mCherry. After infected hemocytes were fixed and stained, they were imaged for DAPI, EGFP, and mCherry using confocal microscopy. Hemocyte infection rates, as determined by the percentage of EGFP-positive cells that also exhibited mCherry signal, for both ex vivo and in vivo C. burnetii infections was nearly 100%. This was expected since an MOI of 10 GE/cell was used for ex vivo infection, which should result in infection of 100% of cells24. Regarding the in vivo infections, since larvae are placed in a high-titer droplet of mCherry expressing-C. burnetii (5.95&#215;109 GE/mL), the number of bacteria is roughly 10,000-fold higher than the number of hemocytes per larva. Therefore, a 100% infection rate is expected for the in vivo infections.
Next, Z-sections were collected through the hemocyte to visualize the entire cell in 3D, and to confirm the presence of C. burnetii in the interior of the cell. Figure 4 shows transparent cross-sections of a hemocyte (in green) with C. burnetii (in red) seen in the interior of the cell. The images were also reconstructed into a 3D model (Figure 5) representing the surfaces of the hemocyte and C. burnetii, again showing C. burnetii in the interior of the cross-sectioned hemocytes. Interestingly, in vivo-infected hemocytes exhibited greater cytoplasmic extensions and were flatter in nature, while ex vivo-infected hemocytes were more spherical (Figure 5). This could indicate a greater population of lamellocytes in the in vivo-infected population and plasmatocytes in the ex vivo population7. While lamellocyte differentiation is generally induced during parasitic wasp infection25, wounding of the Drosophila larvae is also sufficient to induce lamellocyte differentiation26. Finally, while not utilized in the experiments presented here, there are GFP-expressing forms of Listeria27&#160;and IIV628 that could be used to generate 3D models of hemocyte infection. Instead, Listeria- and IIV6-infected hemocytes were used for Western blotting and qRT-PCR experiments.
Using the method presented here, infections are performed both ex vivo and in vivo for imaging. In addition, pathogen mRNA and protein analysis followed ex vivo infections. Specifically, extracted hemocytes were infected with IIV6 and were applied to qRT-PCR analysis. It showed significantly higher levels of IIV6-193R, a viral gene that encodes for a putative inhibitor of apoptosis29, between mock- and IIV6-infected cells (Figure 8A). Electrophoresis of the amplified products confirmed the presence of a band for the IIV6-193R gene product in the infected sample, but not the mock-infected sample (Figure 8B). Amplified bands for the RpII endogenous control were found in all samples, and IIV6 infection in S2 cells were performed as a positive control. Since IIV6 infections were performed at an MOI of 1 TCID50/cell, approximately 50% of the cells were expected to be infected, based on the Poisson distribution24.
Total protein from mock- or Listeria-infected hemocytes was collected at 1, 2, and 4 h post-infection and protein concentration was determined by Bicinchoninic acid (BCA) assay. 100% of the cells were expected to be infected ex vivo since the MOI was 10 CFU/cell24. Western blotting confirms the presence of Listeria-derived protein products in the infected hemocytes, with high levels achieved by 4 h post-infection (Figure 9). Listeria inoculum is used as a positive control for the detection of Listeria-specific bands. The presence of actin is shown in the hemocyte samples to confirm levels of protein loading. Taken together, these results indicate that the hemocytes extracted using the method presented here were suitable for both viral and bacterial infection experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57077/57077fig1.jpg" /> 
Figure 1: Outline of equipment and materials used for hemocyte extraction. Equipment was first prepared prior to dissection. A) The pulled glass capillary was inserted into the nanoinjector after being back-filled with mineral oil. B) The fused tip of the glass capillary was broken with forceps to create a 100 &#181;m outer diameter. B&#39;) DHIM was taken up by the capillary to avoid cell contamination and air bubbles were introduced to make a clear distinction between oil and DHIM. C) Larvae are picked from food vials and placed into a cell strainer. C&#39;) Larvae are washed in sterile water and water is removed with a task wipe, D) Larvae are transferred into a microcentrifuge tube and anesthetized with CO2 gas. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57077/57077fig2.jpg" /> 
Figure 2: Timeline and flow chart for the extraction of hemocytes. A) Larvae are placed on their dorsal side prior to opening the cuticle. B) The cuticle is disrupted with fine pointed forceps and the capillary needle. C) The hemolymph is bled onto paraffin film. D) Pools of hemolymph from 20-50 larvae are taken up with the glass capillary and nanoinjector. E) The hemolymph and hemocytes are transferred into a microcentrifuge tube containing 500 &#181;L DHIM to be counted with a hemocytometer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57077/57077fig3.jpg" /> 
Figure 3: In vivo infection. A) Drosophila fruit juice agar plates and yeast paste are used for incubation of in vivo infected larvae. Cuts are made in the agar plate to facilitate larvae longevity (grey arrows). B) A 0.001 mm pointed tungsten needle is attached to forceps using paraffin film. C) The tip of 0.001 mm pointed tungsten needle. D) The larva is pricked with tungsten needle in the pathogen pool on the paraffin film under stereo microscope. E) The pricked larvae are placed onto the agar plate. F) The plate is sealed with paraffin film and kept on moist paper towels in the container until appropriate time. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57077/57077fig4.jpg" /> 
Figure 4: Cross-sections of pathogen invasion into the hemocytes. Sections extracted from 3D confocal scanning of pathogen-infected hemocytes. A, B) The xy-sections shows C. burnetii (in red marked with white arrowheads) in the interior of the hemocyte. The grey arrowheads show the nuclei of hemocytes. A&#39;, A&#34;, B&#39;) Views including yz- and xz-section images show the invasion of C. burnetii into the hemocytes from 2 additional viewing points. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57077/57077fig5.jpg" /> 
Figure 5: 3D models of pathogen invasion into the hemocytes. Reconstructed 3D models of C. burnetii invasion into the hemocytes are generated following ex vivo and in vivo infection. Model can be freely rotated using the software; here 6 viewing points are shown with the hemocyte in green, C. burnetii in red (white arrowheads), and nucleus in blue (grey arrowheads). The cross-section images showing the interior of pathogen-invaded cells are also shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57077/57077fig6.jpg" /> 
Figure 6: The percentage and population of live hemocytes. Hemocytes were extracted from groups of 10 3rd instar larvae using the method of Petraki et al. and the method presented here. A) The percentage of the live hemocytes was calculated for each technique by Trypan blue exclusion assay. B) The total population of hemocytes was compared between the two techniques. The bar graphs represent the mean &#177; standard deviation from N = 10 biological replicates per method of extraction and assay type. P-values are indicated from a two-tailed Student&#39;s T-test assuming unequal variance. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57077/57077fig7.jpg" /> 
Figure 7: Images of extracted hemocytes using the hemolectin driver. Hemocytes were extracted from 80 larvae of w1118;P{w[+mC]=Hml-GAL4.&#916;}2, P{w[+mC]=UAS-2xEGFP}AH2. The promoter for Hml drives GAL4 in circulating hemocytes, which binds UAS to activate EGFP transcription and expression. The hemocytes were fixed and stained with DAPI as previously described20. Hemocytes are mounted on chambered coverslips and imaged by differential interference contract (DIC) and fluorescence microscopy. The blue channel shows the nucleus stained with DAPI and the green channel shows the hemocytes expressing EGFP. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57077/57077fig8.jpg" /> 
Figure 8: qRT-PCR and gel electrophoresis. The expression of the IIV6-193R gene was observed by qRT-PCR only in the IIV6-infected hemocytes. A) 193R gene expression was normalized to the internal control, RpII, and presented as a ratio. The cycle number for the mock-infected hemocytes was arbitrarily set to a maximum cycle number of 40 for analysis since 193R was not detected in these samples. Data represents the mean &#177; standard deviation from N = 3 biological replicates per group. The P-value indicates a two-tailed Student&#39;s T-test assuming unequal variance. B) The qRT-PCR products are shown by agarose gel electrophoresis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/57077/57077fig9.jpg" /> 
Figure 9: Western Blot Analysis. The expression of Listeria antigens and actin in mock- and Listeria-infected hemocytes from 3rd instar Drosophila larvae at 1, 2, and 4 h post-infection (p.i.) are determined by Western blotting. Total protein concentrations were determined by the Bicinchoninic acid (BCA) assay to normalize total amount of protein loaded in each lane of the gel. Inoculum used to infect the hemocytes was used as a positive control sample. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57077/57077fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Average number of larvae 
			for hemocyte extraction</td>
			<td>Average number of total hemocytes&#160;</td>
		</tr>
		<tr>
			<td rowspan="2">hemolymph 
			capillary extraction 
			(this method)</td>
			<td>192.44 (SD: 86.05)</td>
			<td>4.56 x 105 cells (SD: 5.83 x 105)</td>
		</tr>
		<tr>
			<td>(Range: 70 - 390, N = 25 trials)</td>
			<td>(Range:1.20 x 105 - 2.95 x 106 cells)</td>
		</tr>
		<tr>
			<td rowspan="2">individual 
			larvae dissection</td>
			<td>26.67 (SD: 11.55)</td>
			<td>8.33 x 103 cells&#160; (SD: 6.66 x 103)</td>
		</tr>
		<tr>
			<td>(Range: 20 - 40, N = 10 trials)</td>
			<td>(Range: 4.00 x 103 - 1.60 x 104 cells)</td>
		</tr>
		<tr>
			<td>larval 
			capillary extraction*</td>
			<td>N/A*</td>
			<td>N/A*</td>
		</tr>
		<tr>
			<td colspan="3">*hemocytes were unable to be extracted using this method due to clogging of the capillary needle</td>
		</tr>
	</tbody>
</table>
Table 1: Number of dissected larvae and extracted hemocytes using different techniques. The average numbers of dissected larvae and extracted hemocytes were compared between the method presented here and other methods. Our method resulted in collection of higher numbers of larvae and hemocytes. The larval capillary extraction method was also attempted, but hemocytes were unable to be extracted due to clogging of the capillary tip.

Watch this Scientific Journal Video about Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis at JoVE.com

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.7K Views.  Washington State University. Deze methode laat zien hoe om te visualiseren pathogen invasie in insect cellen met driedimensionale (3D) modellen. Hemocytes van Drosophila larven waren besmet met virale of bacteriële pathogenen, ex vivo of in vivo. Besmette hemocytes werden vervolgens vast en gekleurd voor imaging met een confocal microscoop en de daaropvolgende 3D cellulaire wederopbouw.

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

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Biology

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

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.

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.

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

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Larva

Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
This video describes the Drosophila lymph gland&#8212;the primary site of hematopoiesis in the fly larva&#8212;and shows an example protocol to dissect and mount the organ for immunohistochemistry and fluorescent imaging.

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
This video describes ...

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

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