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 up...

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&#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 &#8220;TIMED&#8221; 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>

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 n...

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

Research

JoVE Journal

Immunology and Infection

7.2K 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 ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

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 ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...