an in vivo assay to quantify bacterial phagocytosis in adult drosophila flies

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

Use a micrometer to hold the needle under a microscope, and use number 5 fine-point stainless steel tweezers to break the tip to a 100-micrometer tip diameter. To measure the volume of liquid that will be injected into each fly, load a capillary needle with sterile 5% food coloring in PBS, and expel the liquid onto a drop of mineral oil on a 0.01-millimeter stage micrometer.
Dispense 10 microliters of 1.6mg/ml particles onto a small square of parafilm, and pull the liquid into the needle. Mount the needle into the injector nozzle, and line the anesthetized flies along their designated area on the fly pad, ventral side up, with the heads oriented toward the front of the pad.
Place the vials and corresponding areas on the bench, and inject the flies at the upper corner of the abdomen with five 100-millisecond pumps of liquid to deliver about 10 nanoliters of particles total. Transfer each fly into the appropriate vial as it is injected, noting the time on the vial.
Next, load a new needle with 0.4% trypan blue solution, and set the pneumatic injector to &#34;Gated&#34;, to allow a constant flow of air to push the liquid out of the needle. 30 minutes after the initial injection, inject each fly abdomen with trypan blue until the abdomens are full and distended. Mount the flys on microscope slides with electrical tape, ventral side down, pushing the wings to the side of the fly to secure them to the tape. Then, gently push the head into the tape to ensure that the fly will not move.
Immediately after all of the flies have been secured, image the insects, one at a time, at a 25 or 32 times magnification on an inverted fluorescence microscope attached to a digital camera and computer, focusing on the dorsal vessel of each fly using the computer software for the digital camera. Then, record the exposure time and magnification between experiments.
To quantify the fluorescence, open an appropriate imaging analysis program, and open one image. To measure the fluorescence intensity of the dorsal vessel, draw a polygon around the dorsal vessel, and select &#34;Measure&#34; to record the fluorescence intensity inside the polygon.
To determine the background fluorescence intensity, copy the first polygon, and move it to an area adjacent to the dorsal vessel of each fly. Then, select &#34;Measure&#34;, and record the fluorescence intensity of the background area.
To normalize the dorsal vessel fluorescence by the background fluorescence, after measuring the fluorescence intensities of the rest of the flies, divide the dorsal vessel fluorescence by the background fluorescence, and calculate the average normalized dorsal vessel fluorescence intensity of all the flies in one strain.

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1. Prepare 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 it 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 it at 25 &#176;C.</li>
	<li>Prepare the pneumatic injector (see Table of Materials) by setting the instrument to 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 them into a loop with the adhesive side out, and place them 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&#39;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. 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 it 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 &#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&#39;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&#39;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="Figure 1" src="/files/ftp_upload/21972/21972_Equation1.jpg" /> 
		where SD1 is the standard deviation of genotype 1 and SD2 is the standard deviation of genotype 2.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2&#956;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>E. coli (K-12 Strain) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>E13231</td>
			<td>Killed E. coli labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~495/~519 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>E23370</td>
			<td>Killed E. coli labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~590/~617 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Fluorescein conjugate</td>
			<td>Invitrogen</td>
			<td>E2861</td>
			<td>Killed E. coli labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~494/~518 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Killed E. coli 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 E. coli BioParticles Conjugate for Phagocytosis</td>
			<td>Invitrogen</td>
			<td>P35361</td>
			<td>Killed E. coli 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 S. aureus BioParticles Conjugate for Phagocytosis</td>
			<td>Invitrogen</td>
			<td>A10010</td>
			<td>Killed S. aureus 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 &#8211; allowing delivery of sub-nanoliter volumes. The amount of liquid delivered depends on two main variables &#8211; the size of the glass needle opening and the amount of time injection pressure is applied. set the instrument to 100 ms &#34;TIMED&#34; mode.</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>S23371</td>
			<td>Killed S. aureus labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~495/~519 nm)</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>S23372</td>
			<td>Killed S. aureus labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~590/~617 nm)</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Fluorescein conjugate</td>
			<td>Invitrogen</td>
			<td>E2851</td>
			<td>Killed S. aureus 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 (Saccharomyces cerevisiae) 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 (Saccharomyces cerevisiae) 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 (Saccharomyces cerevisiae) 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 (Saccharomyces cerevisiae) 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>

Source: Nazario-Toole, A. E., et al. <a href="https://app.jove.com/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.

1. Prepare Fluorescein particles for injection

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

Take anesthetized adult Drosophila flies with their ventral side up.
Load a glass microneedle with heat-killed bacteria, surface-labeled with a fluorophore, suspended in colored liquid to aid visibility during the injection.
Inject the suspension into the upper corner of the abdomen, releasing bacteria into the hemolymph &#8212; the circulatory fluid in the body cavity.
Hemolymph contains immune cells called hemocytes, a subset of which cluster around the abdominal dorsal vessel &#8212; a segment of the circulatory system.
Pattern recognition receptors on hemocytes bind to pathogen-associated molecular patterns in bacteria, causing bacterial phagocytosis.
Rest the flies after the first injection. Then, anesthetize the flies again to inject trypan blue dye into the abdomen.
Under a fluorescence microscope, visualize punctate fluorescence from bacteria phagocytosed by dorsal vessel-associated hemocytes.
Fluorescence emitted by extracellular, non-phagocytosed bacteria is quenched by the injected dye, differentiating them from phagocytosed bacteria.
Obtain the fluorescence intensity ratio of the dorsal vessel to the background, quantifying bacterial phagocytosis.

20 vues. Un test in vivo pour quantifier la phagocytose bactérienne chez les mouches drosophiles adultes

Vidéo: Un test in vivo pour quantifier la phagocytose bactérienne chez les mouches drosophiles adultes - Expérience

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.

Essai de phagocytose pour les cellules apoptotiques dans<em&gt; Drosophila</em&gt; Embryons

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

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Immunologie et infection

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Phagocytose des macrophages alvéolaires et dégagement de bactéries chez la souris

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

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.

Évaluation de la capacité phagocytique spécifique à l’âge des hémocytes adultes de Drosophila melanogaster à l’aide d’un test de phagocytose in vivo

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

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

Transcription

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