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., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+Secretion+of+Lysosomes+at+the+B+Cell+Synapse+Couples+Antigen+Extraction+to+Processing+and+Presentation.">Polarized Secretion of Lysosomes at the B Cell Synapse Couples Antigen Extraction to Processing and Presentation.</a> Immunity. 35 (3), 361-374 (2011).</li><li>Spillane, K. M., Tolar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+antigen+extraction+in+the+B+cell+synapse.">Mechanics of antigen extraction in the B cell synapse.</a> Molecular Immunology. 101, 319-328 (2018).</li><li>Schnyder, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+Cell+Receptor-Mediated+Antigen+Gathering+Requires+Ubiquitin+Ligase+Cbl+and+Adaptors+Grb2+and+Dok-3+to+Recruit+Dynein+to+the+Signaling+Microcluster.">B Cell Receptor-Mediated Antigen Gathering Requires Ubiquitin Ligase Cbl and Adaptors Grb2 and Dok-3 to Recruit Dynein to the Signaling Microcluster.</a> Immunity. , (2011).</li><li>Wang, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+ligand+discrimination+through+a+spreading+and+contraction+response.">B cell ligand discrimination through a spreading and contraction response.</a> Science. 312 (5774), 738-741 (2006).</li><li>Vascotto, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-based+motor+protein+myosin+II+regulates+MHC+class+II+trafficking+and+BCR-driven+antigen+presentation.">The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation.</a> Journal of Cell Biology. 176 (7), 1007-1019 (2007).</li><li>Reversat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+protein+Par3+controls+B-cell+receptor+dynamics+and+antigen+extraction+at+the+immune+synapse.">Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.</a> Molecular biology of the cell. 26 (7), 1273-1285 (2015).</li><li>Lankar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Major+Histocompatibility+Complex+Class+II+Compartments+during+B+Cell+Receptor&#8211;mediated+Cell+Activation.">Dynamics of Major Histocompatibility Complex Class II Compartments during B Cell Receptor&#8211;mediated Cell Activation.</a> The Journal of Experimental Medicine. 195 (4), 461-472 (2002).</li><li>Duchez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+Polarization+of+T+and+B+Cells+at+the+Immunological+Synapse.">Reciprocal Polarization of T and B Cells at the Immunological Synapse.</a> The Journal of Immunology. 187 (9), 4571-4580 (2011).</li><li>P&#233;rez-Montesinos, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+the+intracellular+association+of+selected+rab+small+GTPases+with+MHC+class+II+and+DM+during+dendritic+cell+maturation.">Dynamic changes in the intracellular association of selected rab small GTPases with MHC class II and DM during dendritic cell maturation.</a> Frontiers in Immunology. 8 (MAR), 1-15 (2017).</li><li>Le Roux, D., Lankar, D., Yuseff, M. 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 border="0" cellpadding="0" cellspacing="0" style="width:591px;" width="589">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">IIA1.6 (A20 variant) mouse B-lymphoma cells</td>
			<td style="width:74px;">ATCC</td>
			<td style="width:123px;">TIB-208</td>
			<td style="width:172px;">Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface without Fc&#947;IIR</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">10-mm diameter cover glasses thickness No. 1 circular</td>
			<td>Marienfield-Superior</td>
			<td align="right">111500</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">21985023</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa 488 fluor- donkey ant-rabbit IgG&#160;</td>
			<td>LifeTech&#160;</td>
			<td>A21206</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa Fluor 546 goat anti-Rabbit &#160;IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;A-11071</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Alexa Fluor 647-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;A21238</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Amino- Dynabeads</td>
			<td>ThermoFisher</td>
			<td>14307D</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-pericentrin</td>
			<td>&#160;Abcam</td>
			<td>ab4448&#160;</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-rab6</td>
			<td>Abcam</td>
			<td>ab95954</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Anti-sec61</td>
			<td>Abcam</td>
			<td>ab15575</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BSA</td>
			<td>&#160;Winkler</td>
			<td>&#160;BM-0150</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">CaCl2</td>
			<td>Winkler</td>
			<td>CA-0520</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Culture plate T25</td>
			<td>BD</td>
			<td align="right">353014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fiji Software</td>
			<td style="width:74px;">Fiji col.</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fluoromount G</td>
			<td>Electron Microscopy Science</td>
			<td>17984-25</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">35050061</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutaraldehyde</td>
			<td>Sigma&#160;</td>
			<td>G7651&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glycine</td>
			<td>Winkler&#160;</td>
			<td>BM-0820</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>315-005-003</td>
			<td>IIA1.6 positive ligand</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Goat-anti-mouse IgM antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">31186</td>
			<td>IIA1.6 negative ligand</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HyClone Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30071.03</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">KCl</td>
			<td>Winkler</td>
			<td>PO-1260</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Leica SP8 TCS microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NaCl</td>
			<td>Winkler</td>
			<td>SO-1455</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Nikon Eclipse Ti-E epifluorescence microscope</td>
			<td>&#160;Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Parafilm</td>
			<td>M</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">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 height="19">
			<td height="19" style="height:19px;">Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">15140122</td>
			<td>Liquid</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Polybead Amino Microspheres 3.00&#956;m</td>
			<td>Polyscience</td>
			<td>17145-5</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>Dilute with sterile water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti- alpha tubulin antibody</td>
			<td>Abcam</td>
			<td>ab6160</td>
			<td>1:1000 dilution recommended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti mouse lamp1 antibody</td>
			<td>Cell signaling</td>
			<td align="right">3243</td>
			<td>1:200 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit anti-cep55&#160;</td>
			<td>Abcam</td>
			<td>ab170414</td>
			<td>1:500 dilution recomended but should be optimized</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rabbit Anti-gamma Tubulin antibody</td>
			<td>&#160;Abcam</td>
			<td>ab16504</td>
			<td>&#160;1:1000 for Western Blot</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">RPMI-1640</td>
			<td>Biological Industries</td>
			<td>01-104-1A</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Saponin</td>
			<td>&#160;Merck</td>
			<td align="right">558255</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td align="right">11360070</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sucrose</td>
			<td>Winkler&#160;</td>
			<td>SA-1390&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Triton X-100</td>
			<td>&#160;Merck</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tube 50 ml</td>
			<td>Corning</td>
			<td align="right">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 ...

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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 ...

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Research

JoVE Journal

Immunology and Infection

8.9K Views.  Pontificia Universidad Católica de Chile. 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. 

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Seven Steps to Stellate Cells

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

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


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

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

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

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

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 ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

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 ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...