eoe sub articles

EoE Sub Articles

1. Collect&#160;and Prepare&#160;Flies
<ol>
	<li>Rear&#160;Drosophila melanogaster&#160;under the desired experimental conditions. Take care not to overcrowd the flies during rearing and make sure that larval densities are consistent across treatments since environmental conditions during the larval stage can profoundly affect immune defense phenotypes during the adult stage.</li>
	<li>Collect experimental flies 0 -&#160;3 days after eclosion from the pupal case and transfer them onto fresh medium.</li>
	<li>House the collected flies at a desired temperature (temperatures between 22 &#176;C and 28 &#176;C are generally suitable) until they are aged 5 - 7 days post-eclosion. 
	NOTE: This allows sufficient time for the flies to complete metamorphosis and become mature adults, but is well before senescence begins.</li>
	<li>Sort the desired number of flies into a separate vial before infection, again taking care to avoid overcrowding.&#160;&#160;&#160;&#160;&#160; 
	NOTE: Only males are infected in this demonstration&#160;but is equally possible to infect females using the procedure described.</li>
</ol>
2. Culture&#160;and Prepare&#160;Bacteria
<ol>
	<li>Prepare a master plate of the chosen bacteria at least 2 days before infection. Streak the bacteria from a 15% glycerol stock stored indefinitely at -80 &#176;C. Store the master plate at 4 &#176;C for up to 2 weeks. Always streak the master plates directly from the frozen glycerol stock. Avoid serial passage of bacteria from plate to plate, as repeated passage in culture can cause bacteria to evolve attenuated virulence. 
	NOTE: This example makes use of&#160;Providencia rettgeri&#160;and&#160;Escherichia coli.</li>
	<li>Use the following procedure to prepare a bacterial suspension for injection.
	<ol>
		<li>Grow a 2 ml culture of bacteria by inoculating a sterile medium with a single colony isolated from the master plate. Grow the bacteria to the stationary phase (e.g.&#160;O/N growth at 37 &#176;C).&#160;&#160;&#160;&#160; 
		NOTE: Both&#160;P. rettgeri and E. coli&#160;grow well in Luria Broth at temperatures from 20 - 37 &#176;C with gentle shaking.</li>
		<li>Once the culture has reached the stationary phase, gently pellet cells from approx. 600 &#181;l&#160;of the culture in a tabletop centrifuge (3 min at 5,000 x g), discard the supernatant and resuspend the bacteria in approx. 1,000 &#181;l&#160;of sterile phosphate-buffered saline (PBS; 137 mM Sodium chloride, NaCl, 2.7 mM potassium chloride, KCl, 10 mM sodium phosphate, Na2PO4, 1.8 mM potassium dihydrogen phosphate, KH2PO4, pH = 7.4).</li>
		<li>Dilute the resuspended cells in sterile PBS to achieve an optical density (OD) appropriate for the bacterium being used, measured with a spectrophotometer as the absorbance at 600 nm. Use this suspension to infect the flies.&#160;&#160; 
		NOTE: A600&#160;= 0.1 or 1.0 corresponds respectively to approximately 108&#160;and 109&#160;Providencia rettgeri&#160;or&#160;E. coli&#160;per ml. Because both live and dead bacteria contribute to optical density, it is important to harvest cultures early in the stationary phase before bacterial corpses accumulate and distort the relationship between optical density and the number of viable bacteria introduced during infection.</li>
	</ol>
	</li>
</ol>
3. Infect the Flies
NOTE: As&#160;Drosophila&#160;immunity is influenced by circadian rhythm, it is important to perform infections at a similar time of day across experimental replicates.
<ol>
	<li>Using a Nanoinjector
	<ol>
		<li>Prepare the glass needles for infection.
		<ol>
			<li>Prepare a glass needle by pulling a borosilicate glass capillary using a micropipette puller.</li>
			<li>Using forceps, break off the tip of the needle to create an opening of approximately 50 &#181;m diameter to allow the ejection of liquid.</li>
		</ol>
		</li>
		<li>Assemble the injector.
		<ol>
			<li>Place the sealing O-ring and then the white spacer (large dimple facing outwards) onto the metal plunger.</li>
			<li>Fill the glass needle with mineral oil using a syringe with a 30 G needle.</li>
			<li>Put the filled glass needle through the collet and then place the larger O-ring around its base, about 1mm from the blunt end of the needle.</li>
			<li>Slide the needle onto the metal plunger and gently screw on the collet until secure.</li>
			<li>Eject most of the mineral oil from the injector, but make sure that there is still a small volume of oil in the needle to act as a barrier between the injector and the bacterial suspension. Make sure that there are no air bubbles within the mineral oil or bacterial suspension, or between the two liquids.</li>
			<li>Set the injector to the desired volume for injection (between 9 nl and 50 nl).</li>
		</ol>
		</li>
		<li>Generate&#160;wounding controls.
		<ol>
			<li>Fill the injector needle with sterile PBS by carefully inserting the tip of the capillary needle in a tube of the media and pressing the &#34;fill&#34; button on the injector. 
			NOTE: Sterile bacterial growth media can also be used as wounding control if the experiment requires injection of bacteria suspended in their growth medium. However, because bacterial growth media contains components that may stimulate the immune system or have other effects on the host, an inert carrier such as PBS is preferable.</li>
			<li>Anaesthetize the desired number of flies under a light flow of carbon dioxide (CO2).</li>
			<li>Inject the flies in the anterior abdomen on the ventrolateral surface with the sterile PBS.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			NOTE: It is also possible to inject flies at other sites, such as the sternopleural plate of the thorax, but it is important to keep the injection site consistent within each experiment.</li>
			<li>Place injected flies into fresh vials with a new medium, laying the vials on their side until all of the flies have recovered from the anesthesia to prevent the flies from becoming stuck to the food.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
			NOTE: It is recommended to inject the PBS control flies before injecting bacteria into experimental flies so the same needle can be used for both treatments. It will not always be possible to use the same needle for an entire experiment. In that case, it may be desirable to record which needle was used with which flies and to include needle identity as an experimental factor in statistical analysis.</li>
		</ol>
		</li>
		<li>Inject&#160;the bacterial pathogen.
		<ol>
			<li>Eject the remaining sterile media from the injector and refill the same needle with the bacterial suspension.</li>
			<li>Repeat the above procedure (3.1.3), now injecting the flies with the bacterial suspension prepared in procedure 2.2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Incubator</td>
			<td>Powers Scientific, Inc</td>
			<td>DROS52SD</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Flypads</td>
			<td>FlyStuff</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Airgas</td>
			<td>CD FG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila rearing mix&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Vials polystyrene</td>
			<td>VWR international</td>
			<td>89092-720</td>
			<td></td>
		</tr>
		<tr>
			<td>Nosterile Large Cotton Balls</td>
			<td>Fisher brand</td>
			<td>22-456-883</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lids, Raised Ridge; 100 . x 15 mm;</td>
			<td>VWR international</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar, Miller</td>
			<td>Difco</td>
			<td>244520</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculating Loop</td>
			<td>VWR international</td>
			<td>80094-488</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin Clasic Pipettes in various sizes 
			0.1 &#181;l to 2 &#181;l, 
			2 &#181;l to 20 &#181;l, 
			20 &#181;l to 200 &#181;l, 
			100 &#181;l to 1000</td>
			<td>Rainin</td>
			<td>PR-2 
			PR-20 
			PR-200 
			PR-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (assorted sizes)</td>
			<td>VWR international</td>
			<td>30128-376 
			53503-810 
			16466-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth Base, Miller</td>
			<td>Difco</td>
			<td>241420</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Culture Tubes Borosilicate Glass</td>
			<td>VWR international</td>
			<td>47729-576</td>
			<td></td>
		</tr>
		<tr>
			<td>S-500 Orbital Shaker</td>
			<td>VWR international</td>
			<td>14005-830</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>VWR international</td>
			<td>37001-300</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4 10x</td>
			<td>Invitrogen</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec 3000 Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Semimicrovolume Cuvettes</td>
			<td>Bio-Rad</td>
			<td>223-9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Capillary Puller</td>
			<td>Kopf Needle Pipette Puller</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.5&#39;&#39; Replacement glass Capillaries for Nanojet II</td>
			<td>Drummond Sientific Company</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II</td>
			<td>Drummond Sientific Company</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Syringe</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil, White, light</td>
			<td>Macron Fine Chemicals</td>
			<td>6358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5mL&#160; Microcentrifuge tubes; Seal Rite</td>
			<td>USA Scientific Inc.</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
	</tbody>
</table>

infecting flies with bacterial suspension using a nanoinjector

Source: Khalil, S. et al., <a href="https://app.jove.com/v/52613">Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.</a>&#160;J. Vis. Exp.&#160; (2015)
This video demonstrates the nano-injection of bacterial suspension into the fly body cavity, resulting in bacterial multiplication and infection. The fly&#39;s immune response clears some bacteria, while others escape immune clearance and survive within the host causing an infection.

Infecting Flies with Bacterial Suspension Using a Nanoinjector

Source: Khalil, S. et al., Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster.&#160;J. Vis. Exp.&#160; (2015)
This ...

To infect flies, begin with a narrow-diameter glass needle filled with mineral oil.
Secure the needle onto the nanoinjector plunger with an O-ring and a spacer for precise and controlled injection, ensuring minimal damage to the flies.
Release the mineral oil, leaving a small amount in the needle as a barrier between the bacterial solution and the injector. Load the needle with the bacterial suspension.&#160;
Position an anesthetized Drosophila melanogaster ventrolateral side-up.
Inject the needle into the fly&#39;s anterior abdomen, piercing through the cuticle and epithelial layer.&#160; Deliver the bacterial suspension into the hemocoel &#8212; a body cavity containing hemolymph &#8212; a nutrient-rich circulatory fluid.&#160;
Within the hemocoel, immune cells called hemocytes, circulate alongside the fat body, a specialized tissue equipped with pattern recognition receptors.
Allow the injected flies to recover in a vial containing medium. Injected bacteria utilize the nutrients from hemolymph and multiply, increasing the bacterial load, and establish a systemic infection.
Fat body cell receptors interact with bacterial antigens, triggering an immune response to produce antimicrobial peptides, ultimately eliminating some bacteria.
Circulating hemocytes perform phagocytic activity, engulfing and degrading a few bacteria. However, some bacteria evade the host&#39;s immune response and survive within the flies, establishing a bacterial infection.

infecting-flies-with-bacterial-suspension-using-a-nanoinjector

Research

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

68 Views. Source: Khalil, S. et al., Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster. J. Vis. Exp.  (2015) This video demonstrates the nano-injection of bacterial suspension into the fly body cavity, resulting in bacterial multiplication and infection. The fly's immune response clears some bacteria, while others escape immune clearance and survive within the host causing an infection. 

Video: Infecting Flies with Bacterial Suspension Using a Nanoinjector - Concept

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 ...

JoVE Journal

Immunology and Infection

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 ...

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 ...

Infecting Flies with Bacterial Suspension Using a Nanoinjector

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