Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

This study was, in part or in whole, supported by National Natural Science Foundation of China (31472187 and 31672550) and The Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.

All animal experiments were approved by the local ethics committee of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit Number: SHVRIAU-14-0101).
1. In Vitro Culture of Schistosomes
Note:&#160;Schistosomes represent a biohazard. Workers should wear latex gloves at all times when handling worms, schistosomal suspensions, or other related biological materials. New Zealand rabbits were inf...

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Recent studies on EVs have demonstrated that schistosome EVs play an important role in host-pathogen interactions3,9,12,16. To further address their regulatory functions, it is essential to isolate EVs from schistosomes. Here, we describe an alternative method for SjEV isolation. This method yields a wide range size of SjEVs, from 100 nm to 400 nm, in adult S. japonicum. The following ...

Extracellular vesicles (EVs) are a population of small membrane-bound vesicles encapsulated with various proteins, lipids, and nucleic acids. Recent studies demonstrated that EVs play a crucial role in cell-cell communication, and are involved in the regulation of numerous physiological processes, including cell development, immune regulation, angiogenesis, and cell migration2,3,4,5. Accumulating evidence indicates that EVs, circulating exosomes, and their miRNA cargo represents potential biomarkers of certain diseases6.
Several protozoa such as Trichomonas vaginalis, Trypanosoma cruzi, and Leishmania spp., have been shown to be able to secrete EVs; helminths have additionally been found to secrete EVs into living hosts7. Parasitic EVs have been shown to be involved in the maintenance of infection, pathogenicity8, and immune regulation9. Recent studies in schistosomes both Schistosoma mansoni (S. mansoni) 10,11&#160;and S. japonicum12 have indicated that adult schistosomes secrete exosome-like vesicles that may be involved in the functional regulations of specific biological processes.
To date, several methods have been used to isolate extracellular vesicles, such as ultracentrifugation,ultrafiltration13, the use of polymer-based reagents, size-exclusion chromatography, and immunoaffinity isolation. These different methods possess their own advantages and limitations14. Generally, ultracentrifugation is considered the gold standard method for vesicle isolation. However, this method suffers from the limitation of potential EV aggregation14.
In schistosomes, a couple of methods have been reported for EV isolation: these include ultracentrifugation11,12,10 and the use of commercial EV isolation kit16 for several stages (eggs16, schistosomula11, adult schistosomes10,12,17). Given that microvesicles are of a wide range of size from several hundred nanometers to thousand nanometers, we developed an alternative method that combines with the use of bench centrifuge, ultrafiltration, and a commercial EV isolation kit to isolate the EVs from adult Schistosoma japonicum. The isolated EVs typically possessed a diameter of 100 - 400 nm and were successfully internalized by the recipient cells.

<table border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1160">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:459px;">Material</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:459px;">Total Exosome Isolation Reagent (from cell culture media)</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td style="width:188px;">4478359</td>
			<td style="width:319px;">For isolating S. japonicum extracellular vesicles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">PKH67</td>
			<td style="width:196px;">Sigma-aldrich</td>
			<td style="width:188px;">MINI67</td>
			<td style="width:319px;">For labeling Evs</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">RPMI 1640 Medium</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>11875119</td>
			<td>For parasite culture</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Penicillin-Streptomycin</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">0.22&#956;m syringe filter</td>
			<td style="width:196px;">Merck</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">SnakeSkin Dialysis Tubing</td>
			<td style="width:196px;">Thermo SCIENTIFIC</td>
			<td>68035</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">PEG8000</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>A100159&#160;</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">RIPA</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">EMD Millipore&#8482; Immobilon&#8482; Western Chemiluminescent HRP Substrate (ECL)</td>
			<td style="width:196px;">Fisher scientific</td>
			<td>WBKLS0100&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">DAPI</td>
			<td>Cell Signaling Technology</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">BCA kit</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">CD63 antibodies</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>D160973-0025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">PVDF</td>
			<td>Bio-Rad</td>
			<td>1704273</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Formvar/Carbon 400 mesh</td>
			<td>Ted Pella</td>
			<td>01754-F</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Phosphotungstic Acid</td>
			<td>Ted Pella</td>
			<td>19402</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">anti-mouse IgG-HRP</td>
			<td>CWBIO</td>
			<td>CW0102</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">NCTC clone 1469 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:188px;">ATCC&#174; CCL-9.1&#8482;</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">FBS</td>
			<td style="width:196px;">HyClone</td>
			<td style="width:188px;">SV30087.02</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:459px;">Equipments</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">GE chemoluminescance imaging system</td>
			<td>GE</td>
			<td>ImageQuant LAS4000mini</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Transmission electron microscopy</td>
			<td>Hitachi</td>
			<td>H-7600</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Milli-Q water</td>
			<td>Milli-Q</td>
			<td>Milli-Q Elix</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Eppendor Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5804R</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Zetasizer Nano</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS&#160;</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Ultracentrifuge</td>
			<td style="width:196px;">Beckman</td>
			<td colspan="2">Optima L-100 XP Ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Incubator</td>
			<td style="width:196px;">ESCO</td>
			<td>CCL-107B</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Microscope</td>
			<td style="width:196px;">Zeiss</td>
			<td>Zeiss Axin Observer Z1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

To quantify the yield of isolated SjEVs using the described protocol, we used BCA protein assay to access the protein concentration of the isolated SjEVs from 28-day adult schistosomes. As shown in Table 1, the SjEV protein concentration ranged from 208 &#181;g to 250 &#181;g per 100 mL of medium (Table 1).The particle size, as determined by Malvern nanoparticle analysis, indicated that the isolated SjEVs ranged from 100 nm to 400 nm, with the highest pop...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

The overall goal of this procedure is to isolate extracellular vesicles from the medium of in vitro cultured adult Schistosoma japonicum. This method can efficiently isolate EV in in vitro culture medium from adult Schistosoma japonicum. The main advantage of this technique is able to efficiently isolate extracellular vesicle from short-term in vitro culture medium, which can minimize the stress potentially result in non-physiological EV secretion.Demonstration will be by Juntao Liu, a technician from my lab. Collect the parasites in a 500-milliliter beaker, and wash them thoroughly but gently three times using 200 milliliters of warm PBS. Keep the washes brief, and use gravity sedimentation to separate the parasites.Next, onto 90-millimeter dishes, plate about 200 worm pairs per dish. In the dish, wash the parasites twice more using gravity sedimentation. For these washes, use warm RPMI medium supplemented with pen-strep.Replace the second wash with 40 milliliters of the medium without antibiotics. Then, culture the parasites at 37 degrees Celsius under 5%carbon dioxide for two hours. After two hours, transfer the media from the plates to centrifuge tubes, and then pool the eggs and worm debris at 2, 000 g for 30 minutes at four degrees Celsius.Then, collect and transfer the supernatant to a fresh tube. Next, filter the supernatant through a 0.22-micron syringe filter, and collect the filtered solution into a dialyzing bag with a molecular weight cut-off of 3.5 kilodaltons. Then, dialyze the solution using 8%PEG8000 solution at four degrees Celsius overnight.This step is important for increasing the yield of extracellular vesicles. The next day, transfer the dialyzed solution to a new tube, and add 0.5 volumes of the total exosome isolation reagent. Mix the solution until it is homogeneous, and incubate at about five degrees Celsius overnight.The following morning, centrifuge the mixture at 15, 000 g for one hour at about five degrees Celsius. Next, aspirate and discard the supernatant, and resuspend the pellet of EVs in two milliliters of PBS. The pellet will not likely be visible.For western blot analysis, first determine the protein concentration of the sample, such as with the BCA assay. Then, prepare 10 to 20 micrograms of EVs in 22.5 microliters of RIPA buffer. Next, add 7.5 microliters of 4X loading buffer, and heat the mixture at 95 degrees Celsius for five minutes.After the heat treatment, separate the extracellular vesicle proteins on a 12%SDS-PAGE gel. After running the gel, the proteins are transferred into PVDF membrane. Probe the membrane with anti-CD63 antibodies at a one to 1, 000 dilution.Then, treat the membrane with anti-rabbit IgG-HRP at a one to 5, 000 dilution. Now, use an ECL detection reagent to develop the membrane, and follow with analysis on a chemiluminescence imaging system. Start with transferring five micrograms of sample into a polypropylene ultracentrifuge tube, and adjust the volume to 12 milliliters with RPMI-1640 medium.Then, centrifuge the sample at 110, 000 g for 90 minutes at four degrees Celsius. After the centrifugation, carefully aspirate and discard the supernatant. Then, prepare a 2X extracellular vesicle suspension by adding one milliliter of diluent C to the pellet.Resuspend the pellet with gentle pipetting. Now, prepare fresh 2X dye solution. Add two microliters of the PKH67 ethanolic dye to one milliliter of diluent C in a microfuge tube, and mix well.Then, stain the sample by mixing together the two solutions and incubating the mix at room temperature for four minutes. Next, stop the staining by adding four milliliters of FBS and waiting another minute. Then, add RPMI-1640 medium to adjust the volume to 12 milliliters, and spin down the suspension to isolate the stained vesicles.Remove and discard the supernatant. Then, resuspend the pelleted PKH67-labeled EVs in 12 milliliters of RPMI-1640 medium. Then, repeat the centrifugation, but this time collect the vesicles in one milliliter of PBS.For the uptake assay, prepare overnight cultures of mammalian cells in six-well plates. The next morning, add five micrograms of PKH67-labeled vesicles to each well, and continue the culture for two to four hours. After the incubation, remove the medium, and wash the cells with PBS two times.Then, fix the cells for 15 minutes, wash them twice with PBS, and stain them with DAPI before imaging. Schistosomes at the liver stage were collected from rabbits 28 days post-infection. Extracellular vesicles were then collected from the schistosomes worms and analyzed.Their particle size, as determined by Malvern nanoparticle analysis, ranged between 100 and 400 nanometers, with the bulk of the population near 220 nanometers. Transmission electron microscopy revealed the extracellular vesicles to be round and between 100 and 200 nanometers in diameter. Western blotting analysis indicated that the isolated extracellular vesicles were recognized using CD63 antibodies, which is a typical extracellular vesicle marker.Fluorescence microscopy of PKH67-labeled extracellular vesicles internalized by the NCTC clone 1469 cells showed that the extracellular vesicles were mainly distributed in the cytoplasm of recipient cells. After watching this video, you should have a good understanding of how to isolate EV in in vitro culture medium from adult Schistosoma japonicum.

isolation-characterization-extracellular-vesicles-from-adult

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Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.