The authors acknowledge Lic. Cecilia Guti&#233;rrez Ayesta (Servicio de Microscop&#237;a Electr&#243;nica, CONICET, Bah&#237;a Blanca, Argentina) and Lic. Leonardo Sechi and Lic. Eliana Maza (INIFTA, Universidad Nacional de La Plata, Argentina) for the technical assistance with transmission electron microscopy and dynamic light scattering, respectively. We also thank Dra. Graciela Salerno, Dra. Corina Ber&#243;n and Dr. Gonzalo Cal&#243; for the use of the ultracentrifuge at the INBIOTEC-CONICET-FIBA, Argentina. The authors gratefully acknowledge Lic. Kelly (SENASA, Mar del Plata, Argentina) and Lic. H. N&#250;nez Garc&#237;a (CONICET, Universidad Nacional de Mar del Plata, Argentina) for their collaboration in the welfare assessment of mice, and Med. Vet. J. Reyno, Med. Vet. S. Gonzalez, and Med. Vet L. Netti for their contribution to obtaining parasite material. This work, including the costs of experimentation, reagents, and equipment, was supported by the PICT 2020 No. 1651 financed by the ANPCyT.

The authors declare that they have no conflicts of interest.

The protocol workflow for culturing parasites, isolating parasite-derived sEVs, differentiating dendritic cells from bone marrow, and analyzing sEV uptake by these cells is outlined in Figure 1. The aim was to describe in detail each protocol section that may be carried out as a whole or separately, highlighting the major considerations to guarantee the implementation of the methodology. The analysis of the population of EVs obtained from complete parasitic organisms has a concrete impact on...

Echinococcus granulosus is a zoonotic parasitic helminth responsible for a long-term infection known as cystic echinococcosis1. In intermediate hosts, such as livestock and humans, the parasite infection primarily affects the liver and lungs, where the larval stage develops as fluid-filled cysts or metacestodes containing protoscoleces (a larva itself). Like all cestodes, this parasite lacks both digestive and excretory systems and has, therefore, evolved active endocytic and exocytic cellular processes to regulate the uptake and excretion of metabolites as well as the release of extracellular vesicles2,3. Extracellular vesicles (EVs) are lipid bilayer-enclosed particles secreted by apparently all cell types. In particular, small extracellular vesicles (sEVs), defined as EVs smaller than 200 nm regardless of their biogenesis origin4, can act as intercellular immune mediators. This function is especially significant in parasites, which rely on host immunomodulation to ensure their survival3. Immune manipulation is achieved through the uptake of sEVs by host dendritic cells, the only cells capable of activating naive T cells in vivo and initiating an adaptive immune response that will lead to chronic infection by these parasitic worms. Dendritic cells, professional antigen-presenting cells of the innate immune system, process and load antigenic peptides onto Major Histocompatibility Complex Class I and Class II (MHC I and MHC II) and exhibit them on their membranes for exclusive na&#239;ve T cell priming (CD8+ and CD4+ T cells, respectively)5. Following dendritic cells induce their maturation by induction of expression of the co-stimulatory markers CD80/CD86 and CD40 and MHC-II and migrate from peripheral tissues to secondary lymphoid organs upon recognizing foreign antigens, loading them for exclusive na&#239;ve T cell priming6. Thus, the overall goal of this protocol is to study the helminth parasite-host communication in a realistic manner, analyzing the packaging and delivery of parasitic components in the form of sEVs, which, upon reaching the host immune cells, influence the development of infection and the progression of the chronic parasitic disease.
Addressing the analysis of the helminth-host interface through the study of sEVs has several advantages. First, the tegument, the outer covering of flatworms, is a double membrane structure that constitutes a major crossing point between the parasite and its host, allowing sEVs to be readily generated or permeated from this structure7. Second, sEVs are highly loaded with protein antigens from all stages of the parasite life cycle, representing the natural way through which the host immune system samples antigens during worm infection8,9. Due to their biological production, ease of purification (without requiring tissue disruption or protein fractionation), and direct interaction with host cells, helminth sEVs enable the development of in vitro experiments to simulate the in vivo conditions of parasite-host interaction. Finally, sEVs represent the possibility of having parasitic structures that can be phagocytosed or internalized by host cells, overcoming the impossibility of doing so with whole parasites, particularly in cases of encysted worms.
Considering the advantages mentioned and the fact that helminthiases are prevalent and typically chronic diseases in which parasites presumably manipulate the host immune system as a survival strategy, the isolation of parasite-derived EVs and their study in interaction with dendritic cells provides a valuable framework to explore this immunomodulation10. In this sense, it has been described that the internalization of EVs from helminths, including nematodes and platyhelminths such as Schistosoma mansoni, Fasciola hepatica, Brugia malayi, and E. granulosus, induces the maturation and activation of dendritic cells9,11,12,13,14,15.
The isolation of helminth-derived EVs not only enables the study of immunological interactions, potentially leading to the development of protective vaccines or immunotherapeutic agents for allergic or autoimmune diseases, but also facilitates the exploration of other biological interactions and functions8,16,17. In this context, EVs, which play a role in the natural history of parasitic infections, could be utilized to investigate parasite development and interactions with specific host cells. Moreover, they could have potential applications as early or differential biomarkers for the diagnosis of parasitic diseases, monitoring therapeutic responses, and contributing to the control and management of parasitic infections17,18.
In addition, as previously demonstrated, the larval stage of E. granulosus is susceptible to changes in cytosolic calcium concentration, which, besides playing a role in parasite viability, also controls the exocytosis rate19,20. In this context, and knowing that intracellular calcium elevation enhances EV release, using an intracellular calcium enhancer as loperamide could be a crucial strategy to increase the number of EVs. This approach is particularly interesting for cellular systems that require large populations to generate an adequate quantity of EVs for cargo and functional analysis11,21,22. The current protocol (Figure 1) details the methods for obtaining pure cultures of E. granulosus larval stage and the conditions that enhance sEV production. It also describes the workflow for the isolation and characterization of these vesicles, as well as their uptake by murine dendritic cells, an essential step in the initial study of host immune system modulation.

<table><tbody><tr><td>1.5 mL tubes</td><td>Henso</td><td>N14059</td><td></td></tr><tr><td>24-well plate&amp;nbsp;</td><td>JetBiofil</td><td>CAP011024</td><td>Polystyrene, flat bottom, Sterile</td></tr><tr><td>6-well plate&amp;nbsp;&amp;nbsp;</td><td>Henso Medical Co. Ltd.</td><td>N14221</td><td>Flat-shape bottom, PS material, Sterile</td></tr><tr><td>70 mm polypropylene cell strainer</td><td>Biologix Group Limited</td><td>15-1070</td><td>Sterile</td></tr><tr><td>Alcian blue 8 GX dye&amp;nbsp;</td><td>Santa Cruz</td><td>sc-214517B</td><td></td></tr><tr><td>Automatic CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Nuarire</td><td>UN-5100E/G DH</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Wiener lab</td><td>1443151</td><td></td></tr><tr><td>CD11c Monoclonal antibody-PECy5&amp;nbsp;&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>15-0114-82</td><td>clone (N418)</td></tr><tr><td>CD40 Monoclonal antibody-FITC 100 &amp;micro;g</td><td>eBioscience</td><td>11-0402-82</td><td>clone (HM40-3)</td></tr><tr><td>CD80 Monoclonal antibody-APC 100 &amp;micro;g</td><td>eBioscience</td><td>17-0801-82</td><td>clone (16-10A-1)</td></tr><tr><td>CD86 Monoclonal antibody-FITC&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>11-0862-82</td><td>clone (GL-1)</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>IEC CL31R Multispeed</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>Nikon Confocal Microscope C1</td><td></td></tr><tr><td>Conical tubes 15 mL dia.17 x 120 mm</td><td>Citotest</td><td>4610-1865</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>107K4034</td><td></td></tr><tr><td>D-Glucose</td><td>Merk</td><td>1.78343</td><td></td></tr><tr><td>Dimethyl Sulfoxide&amp;nbsp;</td><td>Anedra</td><td>6646</td><td></td></tr><tr><td>Fetal Bovine Serum 500 mL</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>12352207</td><td></td></tr><tr><td>Flow Cytometry System</td><td>BD Biosciences</td><td>BD FACSCanto&amp;trade; II</td><td></td></tr><tr><td>Folded Capillary Zeta Cell&amp;nbsp;</td><td>Malvern Panalytical</td><td>DTS1070</td><td></td></tr><tr><td>Gentamicin sulfate salt</td><td>Sigma</td><td>G1264</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Fluka</td><td>49630</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>11344041</td><td></td></tr><tr><td>Hypodermic&amp;nbsp; needle 21 G x 1&quot;25/8</td><td>Weigao</td><td></td><td>Sterile</td></tr><tr><td>Hypodermic&amp;nbsp; needle 25 G x 5/8&quot;</td><td>Weigao</td><td></td><td>Sterile</td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Leica</td><td>DMIL LED Fluo&amp;nbsp;</td><td></td></tr><tr><td>Ketamine</td><td>Holliday</td><td></td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp; 5 mg</td><td>Invitrogen</td><td>tlrl-rslps</td><td>LPS from the Gram-negative bacteria E. coli K12 . TLR2/4 Agonists</td></tr><tr><td>Loperamide hydrochloride</td><td>Sigma-Aldrich</td><td>5.08162</td><td></td></tr><tr><td>Medium 199&amp;nbsp;</td><td>Gibco</td><td>11150059</td><td></td></tr><tr><td>Methylene Blue</td><td>Anedra</td><td>6337</td><td></td></tr><tr><td>MHC class I (H2kb) Monoclonal antibody-PE 100 &amp;micro;g</td><td>eBioscience</td><td>12-5958-82</td><td>clone (AF6-88.5.5.3)</td></tr><tr><td>MHC class II (IA/IE) Monoclonal antibody-FITC&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>11-5321-82</td><td>clone (M5/114.15.2)</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CX31</td><td></td></tr><tr><td>Mouse recombinant murine Flt3L.</td><td>PrepoTech</td><td>250-31L-10UG</td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>ND-One</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Agar Scientific</td><td>R1018</td><td></td></tr><tr><td>Penicillin G sodium salt</td><td>Sigma</td><td>P3032-10MU</td><td></td></tr><tr><td>PKH26</td><td>Sigma-Aldrich</td><td>MINI26</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Timper</td><td></td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>RBC lysis buffer 100 mL</td><td>Roche</td><td>11814389001</td><td></td></tr><tr><td>RPMI medium&amp;nbsp; 1640 1x 500 mL</td><td>Sigma-Aldrich (Gibco)</td><td>11875093</td><td></td></tr><tr><td>Sodium Cacodylate&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sodium Chloride</td><td>Anedra</td><td>7647-14-5</td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>Sodium Phosphate Dibasic (Anhydrous) p. a.</td><td>Biopack</td><td>1639.07</td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>Streptomycin sulfate salt</td><td>Sigma</td><td>S9137</td><td></td></tr><tr><td>Syringe 10 mL</td><td>Bremen</td><td></td><td>Sterile</td></tr><tr><td>Thickwall polycarbonate tubes</td><td>Beckman Coulter</td><td></td><td>13 x 55 mm , nominal capacity 4 mL</td></tr><tr><td>Transmission Electron Microscope</td><td>Jeol</td><td>JEOL JSM 100CX II</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima LE-80k&amp;nbsp;</td><td>90 Ti rotor</td></tr><tr><td>Xylazine</td><td>Richmond</td><td></td><td></td></tr><tr><td>Zetasizer Nano&amp;nbsp;</td><td>Malvern&amp;nbsp;</td><td>Nano ZSizer-ZEN3600</td><td>To perform Dynamic Light scattering and zeta potential measurements</td></tr></tbody></table>

isolation and dendritic cell-uptake of small extracellular vesicles from echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

A flowchart summarizing the main steps for maintaining pure cultures of the E. granulosus larval stage, the isolation and characterization of sEVs, and their uptake by murine dendritic cells are shown in Figure 1. To achieve high sEV production from E. granulosus protoscoleces and metacestodes, an in vitro culture method previously developed in the laboratory was employed to maximize the survival and metabolic homeostasis of the studied parasite (<strong class="xfi...

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

isolation-dendritic-cell-uptake-small-extracellular-vesicles-from

Research

JoVE Journal

Immunology and Infection

144 Views.  Universidad Nacional de Mar del Plata (UNMdP). Hier beschrijven we in vitro kweekomstandigheden, isolatie en verhoogde aanmaak van extracellulaire blaasjes (EV's) van Echinococcus granulosus. De kleine EV's werden gekenmerkt door dynamische lichtverstrooiing en transmissie-elektronenmicroscopie. De opname door beenmerg-afgeleide dendritische cellen en hun fenotypische modulatie werden bestudeerd met behulp van confocale microscopie en flowcytometrie.

Video: Isolatie en dendritische celopname van kleine extracellulaire blaasjes van Echinococcus granulosus

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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.

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.

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

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

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