M.-I.Y. is supported by a research grant from FONDECYT #1180900. J.I., D.F. and J.L. were supported by fellowships from the Comisi&#243;n Nacional de Ciencia y Tecnolog&#237;a. We thank to David Osorio from Pontificia Universidad Cat&#243;lica de Chile for the video recording and editing.

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I., Vascotto, F., Yokozeki, T., Faure-Andre&#180;, G., Mougneau, E., Glaichenhaus, N., Manoury, B., Bonnerot, C., Lennon-Dume&#180;nil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syk-dependent+Actin+Dynamics+Regulate+Endocytic+Trafficking+and+Processing+of+Antigens+Internalized+through+the+B-Cell+Receptor.">Syk-dependent Actin Dynamics Regulate Endocytic Trafficking and Processing of Antigens Internalized through the B-Cell Receptor.</a> Molecular biology of the cell. 18 (September), 3451-3462 (2007).</li><li>Reber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8+Isolation+of+Centrosomes+from+Cultured+Cells.">Chapter 8 Isolation of Centrosomes from Cultured Cells.</a> Methods in Molecular Biology. 777 (2), 107-116 (2011).</li><li>Gogendeau, D., Guichard, P., Tassin, A. 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The authors have nothing to disclose.

We describe a comprehensive method to study how B lymphocytes re-organize their intracellular architecture to promote the formation of an IS. This study includes the use of imaging techniques to quantify the intracellular distribution of organelles, such as centrosome, Golgi apparatus and lysosomes during B cell activation, and how they polarize to the IS. Additionally, we describe a biochemical approach to study changes in the centrosome composition upon B cell activation.
To promote the form...

B lymphocytes are an essential part of the adaptive immune system responsible for producing antibodies against different threats and invading pathogens. The efficiency of antibody production is determined by the ability of B cells to acquire, process and present antigens encountered either in a soluble or surface-tethered form1,2. Recognition of antigens attached to the surface of a presenting cell, by the BCR, leads to the formation of a close intercellular contact termed IS3,4. Within this dynamic platform both BCR-dependent downstream signaling and internalization of antigens into endo-lysosome compartments takes place. Uptaken antigens are processed and assembled onto MHC-II molecules and subsequently presented to T lymphocytes. Productive B-T interactions, termed B-T cell cooperation, allow B lymphocytes to receive the appropriate signals, which promote their differentiation into antibody-producing plasma cells or memory cells8.
Two mechanisms have been involved in antigen extraction by B cells. The first one relies on the secretion of proteases originating from lysosomes which undergo recruitment and fusion at the synaptic cleft5,6. The second one, depends on Myosin IIA-mediated pulling forces that triggers the invagination of antigen containing membranes which are internalized into clathrin-coated pits7. The mode of antigen extraction relies on the physical properties of the membrane in which antigens are found. Nevertheless, in both cases, B cells undergo two major remodeling events: actin cytoskeleton reorganization and polarization of organelles to the IS. Actin cytoskeleton remodeling involves an initial spreading stage, where actin-dependent protrusions at the synaptic membrane increase the surface in contact with the antigen. This is followed by a contraction phase, where BCRs coupled with antigens are concentrated at the center of the IS by the concerted action of molecular motors and actin cytoskeleton remodeling8,9,10,11. Polarization of organelles also relies on the remodeling of actin cytoskeleton. For instance, the centrosome becomes uncoupled from the nucleus, by local depolymerization of associated actin, which allows the repositioning of this organelle to the IS5,12. In B cells, repositioning of the centrosome to one cell pole (IS) guides lysosome recruitment to the synaptic membrane, which upon secretion can facilitate the extraction and/or processing of surface-tethered antigens6. Lysosomes recruited at the IS are enriched with MHC-II, which favors the formation of peptide-MHC-II complexes in endosomal compartments to be presented to T cells13. Additionally, the Golgi apparatus has also been observed to be closely recruited to the IS14, suggesting that Golgi-derived vesicles from the secretory pathway could be involved in antigen extraction and/or processing.
Altogether, intracellular organelle and cytoskeleton rearrangements in B cells&#160;during synapse formation are the key steps that allow efficient antigen acquisition and processing required for their further activation. In this work we introduce detailed protocols on how to perform imaging and biochemical analysis in B cells to study the intracellular remodeling of organelles associated with the formation of an IS. These techniques include: (i) Immunofluorescence and image analysis of B cells activated with antigen-coated beads and on antigen-coated coverslips, which allows visualization and quantification of intracellular components that are mobilized to the IS and (ii) isolation of centrosome-enriched fractions in B cells by ultracentrifugation on sucrose gradients, which allows the identification of proteins associated with the centrosome, potentially involved in regulating&#160;cell polarity.

<table><tbody><tr><td>IIA1.6 (A20 variant) mouse B-lymphoma cells</td><td>ATCC</td><td>TIB-208</td><td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface without Fc&amp;gamma;IIR</td></tr><tr><td>100% methanol</td><td>Fisher Scientific</td><td>A412-4</td><td></td></tr><tr><td>10-mm diameter cover glasses thickness No. 1 circular</td><td>Marienfield-Superior</td><td>111500</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>21985023</td><td></td></tr><tr><td>Alexa 488 fluor- donkey ant-rabbit IgG&amp;nbsp;</td><td>LifeTech&amp;nbsp;</td><td>A21206</td><td>1:500 dilution recomended but should be optimized</td></tr><tr><td>Alexa Fluor 546 goat anti-Rabbit &amp;nbsp;IgG</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;A-11071</td><td>1:500 dilution recomended but should be optimized</td></tr><tr><td>Alexa Fluor 647-conjugated phalloidin</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;A21238</td><td>1:500 dilution recomended but should be optimized</td></tr><tr><td>Amaxa Nucleofection kit V</td><td>Lonza</td><td>VCA-1003</td><td>Follow the manufacturer&#039;s directions for mixing the transfection reagents with the DNA</td></tr><tr><td>Amaxa Nucleofector model 2b</td><td>Lonza</td><td>AAB-1001</td><td>Program L-013 used</td></tr><tr><td>Amino- Dynabeads</td><td>ThermoFisher</td><td>14307D</td><td></td></tr><tr><td>Anti-pericentrin</td><td>&amp;nbsp;Abcam</td><td>ab4448&amp;nbsp;</td><td>1:200 dilution recomended but should be optimized</td></tr><tr><td>Anti-rab6</td><td>Abcam</td><td>ab95954</td><td>1:200 dilution recomended but should be optimized</td></tr><tr><td>Anti-sec61</td><td>Abcam</td><td>ab15575</td><td>1:200 dilution recomended but should be optimized</td></tr><tr><td>BSA</td><td>&amp;nbsp;Winkler</td><td>&amp;nbsp;BM-0150</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Winkler</td><td>CA-0520</td><td></td></tr><tr><td>Culture plate T25</td><td>BD</td><td>353014</td><td></td></tr><tr><td>Fiji Software</td><td>Fiji col.</td><td></td><td></td></tr><tr><td>Fluoromount G</td><td>Electron Microscopy Science</td><td>17984-25</td><td></td></tr><tr><td>Glutamine</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma&amp;nbsp;</td><td>G7651&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>Winkler&amp;nbsp;</td><td>BM-0820</td><td></td></tr><tr><td>Goat-anti-mouse IgG antibody</td><td>Jackson ImmunoResearch</td><td>315-005-003</td><td>IIA1.6 positive ligand</td></tr><tr><td>Goat-anti-mouse IgM antibody</td><td>Thermo Fisher Scientific</td><td>31186</td><td>IIA1.6 negative ligand</td></tr><tr><td>HyClone Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>SH30071.03</td><td>Heat inactivate at 56 &lt;sup&gt;o&lt;/sup&gt;C for 30 min</td></tr><tr><td>KCl</td><td>Winkler</td><td>PO-1260</td><td></td></tr><tr><td>Leica SP8 TCS microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>NaCl</td><td>Winkler</td><td>SO-1455</td><td></td></tr><tr><td>Nikon Eclipse Ti-E epifluorescence microscope</td><td>&amp;nbsp;Nikon</td><td></td><td></td></tr><tr><td>Parafilm</td><td>M</td><td>P1150-2</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Merck</td><td>30525-89-4</td><td>Dilute to 4% with PBS in a safety cabinet, use at the moment</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Liquid</td></tr><tr><td>Polybead Amino Microspheres 3.00&amp;mu;m</td><td>Polyscience</td><td>17145-5</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P8920</td><td>Dilute with sterile water</td></tr><tr><td>Rabbit anti- alpha tubulin antibody</td><td>Abcam</td><td>ab6160</td><td>1:1000 dilution recommended but should be optimized</td></tr><tr><td>Rabbit anti mouse lamp1 antibody</td><td>Cell signaling</td><td>3243</td><td>1:200 dilution recomended but should be optimized</td></tr><tr><td>Rabbit anti-cep55&amp;nbsp;</td><td>Abcam</td><td>ab170414</td><td>1:500 dilution recomended but should be optimized</td></tr><tr><td>Rabbit Anti-gamma Tubulin antibody</td><td>&amp;nbsp;Abcam</td><td>ab16504</td><td>&amp;nbsp;1:1000 for Western Blot</td></tr><tr><td>RPMI-1640</td><td>Biological Industries</td><td>01-104-1A</td><td></td></tr><tr><td>Saponin</td><td>&amp;nbsp;Merck</td><td>558255</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher Scientific</td><td>11360070</td><td></td></tr><tr><td>Sucrose</td><td>Winkler&amp;nbsp;</td><td>SA-1390&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>&amp;nbsp;Merck</td><td>9036-19-5</td><td></td></tr><tr><td>Tube 50 ml</td><td>Corning</td><td>353043</td><td></td></tr></tbody></table>

studying organelle dynamics in b cells during immune synapse formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

The present article shows how B cells can be activated using immobilized antigen on beads or coverslips to induce the formation of an IS. We provide information on how to identify and quantify the polarization of different organelles by immunofluorescence and how to characterize proteins that undergo dynamic changes in their association to the centrosome, which polarizes to the IS, using a biochemical approach.
The imaging of B ...

Watch this Scientific Journal Video about Studying Organelle Dynamics in B Cells During Immune Synapse Formation at JoVE.com

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

studying-organelle-dynamics-in-b-cells-during-immune-synapse-formation

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JoVE Journal

Immunology and Infection

9.0K Views.  Pontificia Universidad Católica de Chile. In deze review bespreken we zowel beeldvorming als biochemische technieken die worden gebruikt om organellepositionering en -samenstelling te bestuderen, evenals cytoskeletonherschikkingen die worden waargenomen tijdens de vorming van een immunologische synaps. In de beginfase van de actieve verbouwing kunnen B-cellen hun celoppervlak verhogen en de hoeveelheid antigeen BCR-complexen die bij de synaps worden verzameld, maximaliseren. Aan de andere kant kan lokale rekrutering van lysosomen, sa...

Video: Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

Immunological Synapse Formation Between Helper T Cells and Antigen-Presenting Cells

Source: Bello-Gamboa, A. et al., <a href="https://app.jove.com/v/60312">Imaging the Human Immunological Synapse.</a>&#160;J. Vis. Exp.&#160;(2019) This video demonstrates the in vitro study of immunological synapse formation between an antigen-presenting cell and helper T lymphocytes using a fluorescence microscope. T cell receptors of helper T lymphocytes interact with an antigen-MHC-II complex in antigen-presenting cells and form immunological synapses, followed by secretory vesicle movements toward the synapse, which is visualized by fluorescent microscopy.

Source: Bello-Gamboa, A. et al., Imaging the Human Immunological Synapse.&#160;J. Vis. Exp.&#160;(2019) This video demonstrates the in vitro study of ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Organelle Dynamics in B Lymphocytes following Activation with Antigen-Coated Beads

Source: Iba&#241;ez-Vega, J., et al., <a href="https://app.jove.com/v/59621">Studying Organelle Dynamics in B Cells During Immune Synapse Formation.</a> J. Vis. Exp. (2019)
In this video, we describe a method to study the organelle dynamics in B lymphocytes following their activation with antigen-coated microbeads.

Source: Iba&#241;ez-Vega, J., et al., Studying Organelle Dynamics in B Cells During Immune Synapse Formation. J. Vis. Exp. (2019)
In this video, we ...

Immune Response

Host Defense Mechanism

B Cell Activation on an Antigen-Coated Coverslip

Source: Iba&#241;ez-Vega, J., et al., <a href="https://app.jove.com/v/59621">Studying Organelle Dynamics in B Cells During Immune Synapse Formation.</a> J. Vis. Exp. (2019)
This video demonstrates B cell activation in vitro. The process involves antigen interaction with B cell receptors, leading to cytoskeleton remodeling, immune synapse formation, lysosome movement, antigen internalization, processing, and subsequent presentation on MHC molecules for B cell activation.

Source: Iba&#241;ez-Vega, J., et al., Studying Organelle Dynamics in B Cells During Immune Synapse Formation. J. Vis. Exp. (2019)
This video demonstrates ...

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://app.jove.com/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Samenvatting

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