Name: assessing the cellular immune response of the fruit fly, drosophila melanogaster, using an in vivo phagocytosis assay
Uploaded: 2019-04-10T15:00:00.000+00:00
Duration: 5 min 50 s
Description: Watch this Scientific Journal Video about Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay at JoVE.com

The authors thank Dr. Beth Gonzalez and Dr. Aprajita Garg for support in carrying out the in vivo phagocytosis experiments. An NSF UMD ADVANCE Seed Grant and UMD NIH T32 Training Grants, Cell and Molecular Biology (CMB) and the Host-Pathogen Interactions (HPI), funded this work.

1. Prepare&#160;Fluorescein particles for injection
<ol>
	<li>Reconstitute 10 mg of commercially available, heat-killed bacteria particles labeled with fluorescein (see Table of Materials) to a stock concentration of 10 mg/mL by adding 990 &#181;L sterile 1x PBS and 10 &#181;L 50 mM sodium azide. Vortex to mix.
	<ol>
		<li>Divide into single-use 8 &#181;L aliquots in 0.2 mL tubes and store in a dark box at 4 &#176;C to minimize light associated sensitivity. 
		NOTE: Sodium azide preservative is optional and can be omitted if 10 mg/mL stocks are made with 1 mL sterile 1x PBS, aliquoted, and stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Make a 10 mL solution of 5% food coloring in 1x PBS by mixing 500 &#181;L syringe filtered green food coloring and 9.5 mL sterile 1x PBS.</li>
	<li>Wash particles before injection to remove sodium azide. Mix 42 &#181;L sterile 1x PBS and 8 &#181;L of 10 mg/mL in a 1.7 mL tube. Centrifuge at max speed for 2.5 min at room temperature.
	<ol>
		<li>Remove the supernatant, add 50 &#181;L 1x PBS, and centrifuge at max speed for 2.5 min at room temperature.</li>
		<li>Repeat steps 1.3 and 1.3.1 2x, for a total of 3 washes.</li>
		<li>After the final wash, discard the supernatant and re-suspend particles to 1.6 mg/mL in 50 &#181;L of 5% food coloring in 1x PBS.</li>
		<li>Wrap the tube in aluminum foil to protect from light. Store at 4 &#176;C, discard after 1 week.</li>
	</ol>
	</li>
</ol>
2. Prepare the injection station and flies
<ol>
	<li>Prepare the injection pad. To inject up to 4 genotypes of flies at the same time, use laboratory tape to divide a rectangular CO2 fly pad into 4 sections. On the bench near the microscope, designate areas to place the vials once flies have been lined up on the pad (one for each corner of the pad).</li>
	<li>Prepare vials of age-matched, 4-7 days-old, flies for injection. For each strain to be tested, transfer 5 males and 5 females into a fresh, labeled vial of prepared fly food and keep at 25 &#176;C.</li>
	<li>Prepare the pneumatic injector (see Table of Materials) by setting the instrument to a 100 ms (short bursts of gas pressure to expel the liquid &#8211; allowing the delivery of sub-nanoliter volumes) TIMED mode.</li>
	<li>Prepare the microscope slides. Cut 1.5-inch strips of electrical tape, fold into a loop with the adhesive side out, and place onto a labeled microscope slide.</li>
</ol>
3. Prepare glass capillary needles
<ol>
	<li>Pull glass needles (thin wall glass capillaries) using a needle puller.
	<ol>
		<li>Hold the needle under the microscope with a micrometer and break the tip using #5 fine point stainless steel tweezers. 100 &#181;m tips are sufficient to pierce the fly&#8217;s cuticle while minimizing wounding.</li>
		<li>Measure the volume of liquid that will be injected into each fly. Load the needle with sterile 5% food coloring in 1x PBS and expel the liquid onto a drop of mineral oil on a 0.01 mm stage micrometer. 
		NOTE: If the liquid droplet is spherical, the volume in picoliters is calculated as (size)3/1910.30 A needle with a 100 &#181;m diameter will eject ~2 nL in 100 ms.</li>
	</ol>
	</li>
</ol>
4. Inject flies
<ol>
	<li>Pipette 10 &#181;L of 1.6 mg/mL particles onto a small square of parafilm.
	<ol>
		<li>Pull the liquid into the needle and mount in the injector nozzle (see Table of Materials).</li>
		<li>Anesthetize flies with CO2 and line them up in their designated area on the flypad, with the ventral side up and the heads oriented towards the front of the pad. Place vials in corresponding areas on the bench.</li>
		<li>Inject flies at the upper corner of the abdomen with 5, 100 ms pumps of liquid (~10 nL total).</li>
		<li>Transfer the injected flies to the appropriate vials, note the time on the vial. Keep at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Load a new needle with 0.4% Trypan Blue Solution.</li>
	<li>Set the pneumatic injector to GATED, which allows a constant flow of air to push the liquid out of the needle.</li>
	<li>Anesthetize flies after they have rested for 30 min and inject with Trypan Blue until the abdomen is full and distended. 
	NOTE: When examining phagosome maturation with particles labeled with a pH-sensitive dye, allow flies to rest for 1 h and do not inject with trypan blue before mounting flies.</li>
	<li>Mount flies on microscope slides with electrical tape, ventral side down. Push the wings to the side of the fly and secure them to the tape. Also, gently push the head into the tape to ensure that the fly will not move.</li>
	<li>Immediately move to step 5.</li>
</ol>
5. Imaging flies
<ol>
	<li>Image flies, one at a time, at 25x or 32x magnification using an inverted fluorescence microscope attached to a digital camera and computer (see Table of Materials). Focus on the dorsal vessel of the fly using computer software for the digital camera.</li>
	<li>Record the exposure time and magnification between experiments. 
	NOTE: Losing track of genotypes is a potential source of error when photographing multiple strains in a single sitting. To avoid mislabeling flies, make a note of the image number of the first and last fly photograph for each genotype.</li>
</ol>
6. Quantifying and normalizing the fluorescence
<ol>
	<li>Open the software and open one image at a time.
	<ol>
		<li>Measure the fluorescence intensity of the dorsal vessel. Draw a polygon around the dorsal vessel. Select Measure and record the fluorescence intensity inside the polygon.</li>
		<li>Determine the background fluorescence intensity. Copy the first polygon and move it to an area adjacent to the dorsal vessel of the fly. Select Measure and record fluorescence intensity of the background area.</li>
		<li>Normalize the dorsal vessel fluorescence by the background fluorescence: 
		Dorsal vessel&#160;&#247; background.</li>
		<li>Calculate the average normalized dorsal vessel fluorescence intensity of all flies in a strain.</li>
		<li>Repeat the experiment 2 more times.</li>
		<li>Use a Student&#8217;s unpaired t-test to compare the mean relative fluorescence intensities of control flies and test flies from the three experiments. Calculate the effect size using the formula: Cohen&#8217;s d = (M1-M2) &#247; SDpooled, where M1 is the mean of genotype 1 and M2 is the mean of genotype 2 &#38; 
		SDpooled = <img alt="Equation" src="/files/ftp_upload/59543/59543eq1.jpg" /> 
		where SD1 is the standard deviation of genotype 1 and SD2 is the standard deviation of genotype 2.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Commercially available, fluorescently labeled particles are used to assess phagocytosis in general (0.2 &#181;m carboxylate-modified microspheres) or phagocytosis of microbes (fluorescently-labeled heat- or chemically killed bacteria or yeast). To assess phagosome maturation, researchers can select particles labeled with a pH-sensitive dye that fluoresces when pH decreases from neutral to acidic, as in the phagolysosome. Alternatively, to examine the initial steps of phagocytosis, pathogen recognition and uptake, researchers should choose particles labeled with fluorescent tags that fluoresce inside and outside of cells.
Carefully controlled experimental conditions are key to ensure the in vivo phagocytosis assay is carried out in a reproducible manner. First, Drosophila undergoes cellular immunosenescence32, so flies used in the experiment should be age-matched, optimally within a range of 4-7 days old. Second, the period between injections should be consistent. Phagocytosis occurs rapidly, within minutes of the host cell sensing the microbe14. Over the next hour, phagosome maturation takes place, leading to the acidification of the phagosome and destruction of the internalized microbe. Designating a set interval to wait between injections reduces experimental variability and allows comparisons to be made between strains and experiments conducted on different days. We recommend waiting thirty minutes when using fluorescein particles and one hour for the in vivo phagosome maturation assay using particles labeled with pH-sensitive fluorescent dye.
Using the same needle to inject multiple strains ensures that all flies receive the same dose of particles and experimental variability can arise if glass needles break or become clogged. So, setting up the injection station and flies beforehand is key to ensuring that the in vivo phagocytosis assay is carried out quickly and efficiently. Before starting the experiment, measure the volume of liquid expelled by several needles and retain a set of needles of the same size. Thus, if one needle breaks between injections, it can be replaced immediately. Furthermore, clogged needles can lead to false negative results, making it difficult to distinguish between flies whose hemocytes are unable to phagocytose microbes and flies that did not receive fluorescent particles. Dye solutions made with green food coloring, make it easier to track which flies have been injected and to identify issues with clogged needles. Ideally, the experiment should be performed multiple times, with least 6-8 flies per strain in each experiment, and the results of experiments compared to identify outliers. Finally, enough Trypan Blue should be administered to fully quench the fluorescence of extracellular particles so that the background fluorescence is comparable between flies (Figure 1C).
When imaging flies, care should also be taken to obtain clear, consistent images of the dorsal vessel. Black electrical tape is used to create a dark background and flies should be mounted ventral side down, with their wings and heads secured to the adhesive side of the tape. If possible, flies should remain immobilized under CO2 while images are taken. Finally, to limit the possibility of experimental bias, we recommend using a blinded experimental setup; where vials and strains are coded by one researcher and the experiment is carried out and quantified by another researcher.&#160;
The in vivo phagocytosis assay is a useful tool to identify global defects in phagocytosis. However, it does not distinguish between hemocyte phagocytosis efficiency and differences in hemocyte number or location, due to age-related decline or developmental defects.32 Moreover, the procedure does not provide information on differences in the size or phagocytic capacity of individual hemocytes. Thus, once flies with reduced phagocytosis have been identified using the in vivo phagocytosis assay, researchers should carry out follow-up studies, perhaps using alternative particles, in order to discriminate between immune-related phagocytosis defects and general phagocytic defects. Ultimately, determining the molecular mechanisms that underlie phagocytic defects in mutant strains requires an examination of phagocytosis in single hemocytes. This can be accomplished using, for example, confocal microscopy, fluorescence activated cell sorting or magnetic bead isolation of adult hemocytes27,28,29,32,33,34.
Overall, Drosophila has proven to be an effective model to study innate immunity in live animals. Here we have provided an overview of the in vivo phagocytosis assay, a method to study the global phagocytosis and the cellular immune response in adult flies. The procedure can be applied to screen for new genes that may be important for host defense, validate genes identified in large-scale in vitro RNAi screens, and further characterize mutants with defects in the humoral, gut or antiviral immune responses. It is easy to set-up and can yield a large amount of reliable data about multiple strains of flies in a short amount of time.

Innate immune defenses form the first line of defense against pathogenic microbes. These responses can be functionally divided into humoral and cellular innate immunity, both of which are mediated by germline-encoded pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs)1. Many of the signaling pathways and effector mechanisms of innate immunity are conserved in mammals and invertebrates, such as the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster2. The fruit fly has emerged as a powerful system to study host defense against infectious microorganisms3. Drosophila is genetically tractable, easily and inexpensively reared in laboratories, and has a short generation time. Furthermore, the fruit fly exhibits highly efficient defenses against an array of microbes, enabling the examination of host immunity against viral, bacterial, fungal, or parasitic pathogens.
Drosophila immunologists have historically utilized&#160;forward genetic screens, genome-wide RNA-mediated interference (RNAi) screening of insect cell lines, and pre-existing mutant fly strains to examine innate immunity &#8211; leading to the identification and characterization of several evolutionarily conserved humoral immune pathways4,5,6,7,8. The humoral innate immune response is, arguably, the best characterized immune defense in fruit flies. Following infection, the humoral response leads to the production and systemic release of antimicrobial peptide (AMP) molecules into the hemolymph, the blood equivalent in insects. AMPs are produced by highly conserved Toll and Imd signaling pathways. The Toll pathway is homologous to mammalian TLR/IL-1R receptor signaling, and the Imd pathway is homologous to mammalian Tumor necrosis factor-alpha signaling. In Drosophila, Toll signaling is induced by gram-positive bacteria, fungi, and Drosophila X virus6,9,10 and Imd signaling is induced by gram-negative bacteria11,12.
Cellular immunity, comprised of encapsulation, melanization, and phagocytosis of invasive pathogens,&#160;carried out by specialized blood cells called hemocytes13. There are three classes of hemocytes in the fruit fly: crystal cells, lamellocytes, and plasmatocytes13. Crystal cells, which make up 5% of the circulating hemocytes in larvae, release proPhenoloxidase (proPO) enzymes leading to the melanization of pathogens and host tissues at wound sites. Lamellocytes, which are not normally found in healthy embryos or larvae, are adherent cells that encapsulate foreign objects. These cells are induced upon pupariation or when parasitizing wasp eggs are deposited in larvae. Phagocytic plasmatocytes, which make up 95% of circulating hemocytes in larvae and all remaining hemocytes in adults, play a role in tissue remodeling during development and, notably, serve as the main effector cell of Drosophila cellular immunity.
Phagocytosis an immediate and crucial line of innate immune defense; microbes that breach the host epithelial barrier are quickly engulfed and eliminated by phagocytic blood cells (For a comprehensive review of cell biology of phagocytosis see reference 14). This process is initiated when germline-encoded pattern recognition receptors (PRRs) on hemocytes recognize pathogen associated molecular patterns (PAMPs) of microbes. Once bound to their targets, PRRs initiate signaling cascades that lead to the formation of pseudopods through actin cytoskeleton remodeling. The pseudopods surround the microbe, which is subsequently engulfed and internalized into a nascent organelle, the phagosome. Microbes are destroyed as the phagosome undergoes the process of phagosome maturation when the phagosome is trafficked towards the interior of the hemocyte and acidifies through a series of interactions with lysosomes. In vitro and cell biology studies in mammalian primary cells have been instrumental in identifying and characterizing factors that regulate phagocytosis, such as the mammalian Fc-gamma receptor and C3b receptors15,16. Nevertheless, the ability to execute large-scale screens or in vivo studies are limited in mammalian systems.
Here we present an in vivo assay for phagocytosis in adult fruit flies, which is based on a procedure first introduced by the laboratory of David Schneider in 200017. The Schneider lab showed that sessile hemocytes clustered along the abdominal dorsal vessel readily phagocytose polystyrene beads and bacteria. To visualize phagocytosis, flies are injected with fluorescently labeled particles (such as E. coli labeled with fluorescein isothiocyanate (E. coli-FITC)), incubated for 30 minutes to allow hemocytes time to engulf the particles, and then injected with trypan blue, which quenches the fluorescence of particles not phagocytosed during the incubation period. Fly dorsal vessels are then imaged using an inverted fluorescent microscope. This seminal paper, using a relatively simple experiment, demonstrated that hemocytes phagocytose bacteria and latex beads, that bacterial phagocytosis can be inhibited by pre-injecting flies with latex beads, and that flies without both cellular and humoral immune responses are susceptible even to E. coli. The assay presented in this report builds on the work of the Schneider lab to quantify in vivo phagocytosis by measuring the fluorescence intensity of particles engulfed by dorsal vessel associated hemocytes.
Similar to the approach taken in mammalian systems, Drosophila geneticists initially used genome-wide in vitro RNAi screens to identify genes required for the cellular immune response18,19,20,21,22,23. However, the development of the adult in vivo phagocytosis assay enabled follow-up experiments to be readily carried out in whole animals, thus allowing researchers to verify the biological the role of factors identified in in vitro studies. Such was the case with the transmembrane receptor Eater, which was first identified as a bacterial receptor in an RNAi screen using S2 cells24 and then later shown to mediate Escherichia coli (E. coli), Enterococcus faecalis, and Staphylococcus aureus (S. aureus) phagocytosis in adults25.
Our lab employed the in vivo phagocytosis assay in forward genetic screens and genome-wide association studies (using the Drosophila Genetic Reference Panel (DGRP)) to identify novel genes that regulate phagocytosis in adult hemocytes. These studies led to the characterization the receptors PGRP-SC1A and PGRP-SA26, the intracellular vesicle trafficking protein Rab1427, the glutamate transporter Polyphemus28, and RNA-binding protein Fox-129.
We anticipate that future screens incorporating the in vivo phagocytosis could lead to the identification of additional genes that are important for the cellular immune response in Drosophila. Screens using fully-sequenced inbred lines, such as the DGRP or the Drosophila Synthetic Population Resource (DSPR), can identify natural variants affecting phagocytosis or hemocyte development. Furthermore, the technique could be adopted in other species of Drosophila or used to screen new community resources, such as the collection of 250 Drosophila species maintained by the National Drosophila Species Stock Center (NDSSC) at Cornell. These experiments can be carried out using fluorescently-labeled bacterial or fungal-wall bioparticles that are available commercially or may be performed using any number of bacterial or fungal species &#8211; provided that the microbe expresses fluorescent markers.

<table><tbody><tr><td>0.2&amp;mu;m Red Fluorescent Carboxylate Modified FluoSpheres</td><td>Invitrogen</td><td>F8810</td><td>Fluorescently-labeled latex beads to test general phagocytic capacity of phagocytes. (~580/~605 nm) Inject a 1:20 dilution in PBS with 5% dye.</td></tr><tr><td>5430-10 PicoNozzle Kit</td><td>World Precision Instruments</td><td>5430-10</td><td>Holder for 1.0mm pipette</td></tr><tr><td>&lt;em&gt;E. coli (&lt;/em&gt;K-12 Strain) BioParticles, Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>E13231</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~495/~519 nm)</td></tr><tr><td>&lt;em&gt;E. coli (&lt;/em&gt;K-12 Strain) BioParticles, Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>E23370</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~590/~617 nm)</td></tr><tr><td>&lt;em&gt;E. coli (&lt;/em&gt;K-12 Strain) BioParticles, Fluorescein conjugate</td><td>Invitrogen</td><td>E2861</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~494/~518 nm)</td></tr><tr><td>&lt;em&gt;E. coli (&lt;/em&gt;K-12 Strain) BioParticles, Texas Red conjugate</td><td>Invitrogen</td><td>E2863</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)</td></tr><tr><td>&lt;em&gt;E. coli (&lt;/em&gt;K-12 Strain) BioParticles, Texas Red conjugate</td><td>Invitrogen</td><td>E2863</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)</td></tr><tr><td>Needle Pipette Puller</td><td>David Kopf Instruments</td><td>Model 725</td><td></td></tr><tr><td>pHrodo Red&lt;em&gt; E. coli &lt;/em&gt;BioParticles Conjugate for Phagocytosis</td><td>Invitrogen</td><td>P35361</td><td>Killed &lt;em&gt;E. coli&lt;/em&gt; labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-negative bacteria. (~560/~585 nm). No need to quench with Trypan Blue.</td></tr><tr><td>pHrodo Red&lt;em&gt; S. aureus &lt;/em&gt;BioParticles Conjugate for Phagocytosis</td><td>Invitrogen</td><td>A10010</td><td>Killed &lt;em&gt;S. aureus&lt;/em&gt; labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-positve bacteria. (~560/~585 nm). No need to quench with Trypan Blue.</td></tr><tr><td>Pneumatic PicoPump PV820</td><td>World Precision Instruments</td><td>SYS-PV820</td><td>The World Precision Instruments Pneumatic PicoPump PV820 uses differential pressures to hold liquid in the glass needle between injections. The user manually controls short bursts of gas pressure to expel the liquid &amp;ndash; allowing delivery of sub-nanoliter volumes. The amount of liquid delivered depends on two main variables &amp;ndash; the size of the glass needle opening and the amount of time injection pressure is applied. set the instrument to 100 ms &amp;ldquo;TIMED&amp;rdquo; mode.</td></tr><tr><td>&lt;em&gt;S. aureus &lt;/em&gt;(Wood Strain without protein A) BioParticles, Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>S23371</td><td>Killed &lt;em&gt;S. aureus&lt;/em&gt; labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~495/~519 nm)</td></tr><tr><td>&lt;em&gt;S. aureus &lt;/em&gt;(Wood Strain without protein A) BioParticles, Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>S23372</td><td>Killed &lt;em&gt;S. aureus&lt;/em&gt; labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~590/~617 nm)</td></tr><tr><td>&lt;em&gt;S. aureus &lt;/em&gt;(Wood Strain without protein A) BioParticles, Fluorescein conjugate</td><td>Invitrogen</td><td>E2851</td><td>Killed &lt;em&gt;S. aureus&lt;/em&gt; labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~494/~518 nm)</td></tr><tr><td>Thin Wall Glass Capillaries</td><td>World Precision Instruments</td><td>TW100F-3</td><td>Needles for injection. OD = 1.0 mm</td></tr><tr><td>Trypan Blue Solution (0.4%)</td><td>Sigma</td><td>T8154</td><td>Used to quench extracellular fluorescence of Fluorescein, Alexa Fluor, or Texas Red labeled particles.</td></tr><tr><td>ZEISS SteREO Microscope (Discovery.V8)</td><td>Zeiss</td><td>SteREO Discovery.V8</td><td>Inverted fluorescence microscope for imaging flies. Use a digital camera (example: AxioCam HC camera) and the accompanying software (example: AxioVision 4.7 software) to take pictures.</td></tr><tr><td>Zymosan A (&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;) BioParticles, Alexa Fluor 488 conjugate</td><td>Invitrogen</td><td>Z23373</td><td>Killed labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of yeast. (~495/~519 nm)</td></tr><tr><td>Zymosan A (&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;) BioParticles, Alexa Fluor 594 conjugate</td><td>Invitrogen</td><td>Z23374</td><td>Killed labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of yeast. (~590/~617 nm)</td></tr><tr><td>Zymosan A (&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;) BioParticles, Fluorescein conjugate</td><td>Invitrogen</td><td>Z2841</td><td>Killed labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of yeast. (~494/~518 nm)</td></tr><tr><td>Zymosan A (&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;) BioParticles, Texas Red</td><td>Invitrogen</td><td>Z2843</td><td>Killed labeled with Texas Red. Use to test phagocyte recogntion and uptake of yeast. (~595/~615 nm)</td></tr></tbody></table>

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.

A schematic of the in vivo phagocytosis assay using fluorescein-labeled particles is shown in Figure 1A. Flies are mounted ventral side down on a piece of electrical tape and the first two segments of the abdomen, where the dorsal vessel is located, is clearly visible (Figure 1B). Key sources of experimental error arise at the injection and imaging steps of the procedure (Figure 1C). Using the same needle to inject multiple flies may cause it to become clogged with fly tissue or particles (top left panel in Figure 1C). Because Drosophila auto-fluoresces under GFP light, flies injected with a clogged needle will fluoresce faintly but lack the clear, punctate fluorescence of particles internalized by hemocytes. Another potential source of experimental error can occur during the Trypan Blue injections. Thirty minutes after the first injection with fluorescently-labeled particles, flies receive a second injection with Trypan Blue, which quenches the fluorescence of extracellular, un-phagocytosed particles. Relative fluorescence intensity for each fly is calculated by dividing the fluorescence of the dorsal vessel by that of an equally sized adjacent area on the abdomen. Flies that do not receive enough Trypan Blue fluoresce brightly throughout their entire abdomen (bottom left panel of Figure 1C). This bright fluorescence can reduce the ratio of the dorsal vessel to background fluorescence, thus decreasing the true fluorescence intensity ratio of the animal. Finally, flies should be photographed while immobilized by CO2, as actively moving flies produce blurry images that cannot be quantified (top and bottom, right panels of Figure 1C).
The Zuker collection is a panel of ethyl methanesulfonate (EMS) treated flies with autosomal mutations.31 Originally established as a community resource in 2004, the lines have been used to carry out forward and reverse genetic screens. Our lab identified a line from the Zuker collection that is unable to phagocytose gram-negative (E. coli K-12 strain) and gram-positive bacteria (S. aureus Wood strain without protein A) (Figure 2). This mutant, called argus, shows almost no dorsal vessel fluorescence in the in vivo phagocytosis assay. When compared to the Zuker isogenic background strain, cn bw, argus shows significantly less uptake of E. coli (p-value = 0.019; Cohen&#8217;s d = 1.677) and S. aureus (p-value = 0.0083; Cohen&#8217;s d = 1.672). Argus bacterial phagocytosis is also significantly impaired, E. coli (p-value = 0.045; Cohen&#8217;s d = 0.850) and S. aureus (p-value = 0.0136; Cohen&#8217;s d = 1.422) when compared to another common laboratory control strain, Canton-S.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59543/59543fig1.jpg" /> 
Figure 1: Overview and representative images of in vivo phagocytosis assay. A. Schematic of the in vivo phagocytosis assay using fluorescein-labeled particles. Two injections, 30 minutes apart, are used to visualized particle recognition and uptake by dorsal vessel-associated blood cells. B. White light image showing a fly mounted on tape, with clearly visible anterior abdominal segments. C. Example in vivo phagocytosis images. (top left) A fly injected with a clogged needle has no particle fluorescence. (bottom left) A fly with high extracellular fluorescence because of insufficient trypan blue. (top right) An immobilize fly with no extracellular fluorescence &#8211; ideal image. (bottom right) A fly moving as CO2 anesthesia wears off creates a blurry image. Scale bar, 0.2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59543/59543fig1large.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/59543/59543fig2.jpg" /> 
Figure 2: Phagocytosis of bacteria in mutant and control flies. A. Representative images of phagocytosis bacteria labeled with fluorescein dye; E. coli (top panels) and S. aureus (bottom panels). B. The fluorescence intensity ratio of flies injected with fluorescein-E. coli particles. C. The fluorescence intensity ratio of flies injected with fluorescein-S. aureus particles. n = 3 experiments for each type of bacteria, with 6-8 flies per experiment. Error bars, &#177; SEM. Statistical analysis = Two-tailed t-test. Scale bar, 0.2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59543/59543fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay at JoVE.com

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.

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 ...

assessing-cellular-immune-response-fruit-fly-drosophila-melanogaster

Nanyang Technological University

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JoVE Journal

Immunology and Infection

7.3K Views.  University of Maryland. This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

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

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

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

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

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

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 ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

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

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

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

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

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

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 ...

Biology

Injection of Adult Drosophila Flies: A Method of Compound or Label Delivery

Source: Nazario-Toole, A. E., Wu, L. P. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59543/" target="_blank">Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay</a>. J. Vis. Exp. (2019).
This video describes how to perform an injection of live adult flies and includes an example protocol in which labeled particles and trypan blue are injected in fly abdomens to assay phagocytosis.

Wstrzyknięcie dorosłych much Drosophila: metoda dostarczania związku lub etykiety

Source: Nazario-Toole, A. E., Wu, L. P. Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

Source: Nazario-Toole, A. E., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59543">Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay.</a> J. Vis. Exp. (2019)
This video demonstrates an in vivo assay designed to measure bacterial phagocytosis in adult Drosophila flies. Following the injection of fluorescently labeled bacteria into the abdomen of adult flies, hemocytes associated with the dorsal vessel engage in phagocytosis. The introduction of trypan blue dye serves to quench the fluorescence emitted by extracellular, non-phagocytosed bacteria. This quenching process enhances the identification of phagocytosed bacteria when observed under a fluorescence microscope.

Test in vivo do ilościowego określenia fagocytozy bakteryjnej u dorosłych much Drosophila

1. Prepare Fluorescein particles for injection

	Reconstitute 10 mg of commercially available, heat-killed bacteria particles labeled with fluorescein ...

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

The Phagocytosis Assay: An Immunostaining Technique to Visualize Apoptotic Cell Engulfment in the Embryonic Cells of Drosophila melanogaster

To immunostain the cells with anti-Croquemort antibody, first, serially soak the slide in methanol, then PBS supplemented with 0.2% Triton X-100, followed by PBS alone. Next, block the non-specific binding with 20 microliters of 5% whole swine serum in PBS + 0.2% Triton X-100, for 20 minutes at room temperature. Remove the excess blocking solution, and label the cells with 20 microliters of anti-Croquemort antiserum at 4 degrees Celsius overnight.
The next morning, wash the slide in PBS + Triton X-100 for five 10-minute incubations. After the last wash, rinse the slide in PBS for 10 minutes. Then, label the cells with 20 microliters of alkaline phosphatase-labeled anti-rat IgG at room temperature for 1 hour.
At the end of the incubation, wash the slide five times in PBS + Triton X-100 as demonstrated, followed by a 10-minute soak in buffer solution. Then, label the cells with phosphatase substrate solution, and observe the cells under a light microscope.
When strong purple signals appear in the hemocyte granules, remove the excess substrate, and soak the slide in buffer supplemented with EDTA for 5 to 10 minutes. At the end of the incubation, rinse the cells with two 5-minute PBS washes, and treat them with equilibration buffer for 10 minutes.
After removing the solution, label the cells with 20 microliters of terminal deoxynucleotidyl transferase solution at 37 degrees Celsius for 1 hour. Then, remove the solution from the slide, and soak the cells in 0.5 milliliters stop wash buffer in 17 milliliters of water, for 10 minutes. Next, rinse the cells with three 5-minute PBS washes, and label them with 20 microliters of anti-digoxigenin peroxidase for 30 minutes at room temperature.
At the end of the incubation, wash the cells four times in PBS, as demonstrated, and soak the slide in peroxidase substrate for 30-second increments until the apoptotic cells turn brown. When an optimal staining has been achieved, soak the slide in water to stop the peroxidase reaction.

Test fagocytozy: technika barwienia immunologicznego do wizualizacji pochłaniania komórek apoptotycznych w komórkach embrionalnych Drosophila melanogaster

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

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

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