Name: real-time live imaging of t-cell signaling complex formation
Uploaded: 2013-06-23T13:00:00
Description: Watch this Scientific Journal Video about Real-time Live Imaging of T-cell Signaling Complex Formation at JoVE.com

De auteurs danken Sophia Fried voor technische ondersteuning. MBS bedankt de volgende organisaties voor hun onderzoek support: The Israel Science Foundation voor subsidies no.1659/08, 971/08, 1503-1508 en 491/10, de ministeries van Volksgezondheid en Wetenschap voor subsidies niet. 3-4114 en 3-6540, het Israël Cancer Association door de Estate van wijlen Alexander Smidoda, en de Taubenblatt Family Foundation voor de Bio-medische excellentie subsidie.

1. T-cel transfectie <ol><li> Bereid de geactiveerde Amaxa Nucleofector oplossing door het mengen van de kit &quot;Supplement&quot; oplossing voor het Nucleofector Solution. De geactiveerde oplossing kan bij 4 ° C gedurende drie maanden opgeslagen. </li><li> Kweek Jurkat T-cellen in kweekmedium [10% foetaal kalf serum (FCS), 1% penicilline-streptomycine-oplossing en 2 mM L-glutamine in RPMI]. Optimaal dienen cellen worden 1-2 weken na ontdooien. Met behulp van celculturen in logaritmische groei is cruciaal voor hoge ...

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Immunol. 206, 1-14 (1996).</li><li>Jager, D., Jager, E., Knuth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+tumour+antigens:+implications+for+antigen+specific+immunotherapy+of+cancer.">Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer.</a> J. Clin. Pathol. 54, 669-674 (2001).</li><li>Davis, M. M., Bjorkman, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+antigen+receptor+genes+and+T-cell+recognition.">T-cell antigen receptor genes and T-cell recognition.</a> Nature. 334, 395-402 (1988).</li><li>Burkhardt, J. K., Carrizosa, E., Shaffer, M. 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Immunol. 26, 233-259 (2008).</li><li>Reicher, B., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+leading+from+the+T-cell+antigen+receptor+to+the+actin+cytoskeleton+network.">Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network.</a> FEBS Lett. 584, 4858-4864 (2010).</li><li>Barda-Saad, M., 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+molecular+interactions+linking+the+T+cell+antigen+receptor+to+the+actin+cytoskeleton.">Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton.</a> Nat. Immunol. 6, 80-89 (2005).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+a+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296, 913-916 (2002).</li><li>Adler, J., Parmryd, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+colocalization+by+correlation:+the+Pearson+correlation+coefficient+is+superior+to+the+Mander's+overlap+coefficient.">Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander's overlap coefficient.</a> Cytometry A. 77, 733-742 (2010).</li><li>Costes, S. V., 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=Automatic+and+quantitative+measurement+of+protein-protein+colocalization+in+live+cells.">Automatic and quantitative measurement of protein-protein colocalization in live cells.</a> Biophys. J. 86, 3993-4003 (2004).</li><li>Fang, N., Motto, D. G., Ross, S. E., Koretzky, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosines+113,+128,+and+145+of+SLP-76+are+required+for+optimal+augmentation+of+NFAT+promoter+activity.">Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity.</a> J. Immunol. 157, 3769-3773 (1996).</li><li>Wunderlich, L., Farag&#243;, A., Downward, J., Buday, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Nck+with+tyrosine-phosphorylated+SLP-76+in+activated+T+lymphocytes.">Association of Nck with tyrosine-phosphorylated SLP-76 in activated T lymphocytes.</a> Eur. J. Immunol. 29, 1068-1075 (1999).</li><li>Pauker, M. H., Reicher, B., Fried, S., Perl, O., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cooperation+between+the+proteins+Nck+and+ADAP+is+fundamental+for+actin+reorganization.">Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization.</a> Mol. Cell Biol. 31, 2653-2666 (2011).</li><li>Barda-Saad, M., 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=Cooperative+interactions+at+the+SLP-76+complex+are+critical+for+actin+polymerization.">Cooperative interactions at the SLP-76 complex are critical for actin polymerization.</a> EMBO J. 29, 2315-2328 (2010).</li><li>Pauker, M. H., Hassan, N., Noy, E., Reicher, B., Barda-Saad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color. 5, rs3 (2012).</li></ol>

De regulatie en werking van verschillende cellulaire processen zijn afhankelijk van de vorming en de beëindiging van eiwit-eiwit interacties. Microscopische beeldvorming maakt de real-time tracking van fluorescent gelabelde eiwitten in levende cellen. Colocalization van gemerkte eiwitten suggereren een directe of indirecte interactie tussen de eiwitten en kunnen worden gebruikt om de verkregen resultaten van biochemische werkwijzen, zoals immunoprecipitatie versterken. In tegenstelling tot de biochemische methoden, lee...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amaxa human T Cell nucleofector kit</td>
			<td>Lonza</td>
			<td>VCA-1002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti CD3 (UCHT1 clone)</td>
			<td>BioLegend</td>
			<td>300432</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Falcon FACS Tubes</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>HyClone</td>
			<td>SV30160.03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Calbiochem</td>
			<td>345810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>German coverglass system 4 chamber slides</td>
			<td>Lab-Tek II</td>
			<td>155382</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Bio Lab</td>
			<td>8410501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Biological Industries</td>
			<td>03-025-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hygromycin</td>
			<td>Enzo</td>
			<td>ALX-380-306</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.1% (w/v) in H2O</td>
			<td>Sigma</td>
			<td>P8920</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Accublock digital dry bath</td>
			<td>Labnet</td>
			<td>D1105A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5810 R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510 Meta</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heatable mounting frame - Heating Insert P S</td>
			<td>PeCon</td>
			<td>130-800-031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TempModule S (required for the heatable mounting frame)</td>
			<td>Zeiss</td>
			<td>411860-9010-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporation device</td>
			<td>Amaxa</td>
			<td>Nucleofector I</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence activated cell sorter</td>
			<td>Becton Dickinson</td>
			<td>FACSVantage SE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Bitplane</td>
			<td>7.0.0</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time live imaging of t-cell signaling complex formation

Bescherming tegen besmettelijke ziekten gemedieerd door het immuunsysteem 1,2. T-lymfocyten zijn het meestercoördinatoren van het immuunsysteem, het reguleren van de activatie en reacties van meerdere immuuncellen 3,4. T-celactivering is afhankelijk van de herkenning van antigenen weergegeven door antigen presenterende cellen (APCs). De T-cel antigeen receptor (TCR) is voor elke T-cel kloon en bepaalt antigeenspecificiteit 5. De binding van de TCR aan het antigeen induceert fosforylatie van componenten van het TCR complex. Om T-cel activatie te bevorderen, moet dit signaal worden getransduceerd door het membraan naar het cytoplasma en de nucleus, inleiding diverse belangrijke reacties zoals recrutering signaaleiwitten de TCR, APC site (de immune synaps), hun moleculaire activatie, cytoskelet omlegging, verhoging van de intracellulaire calciumconcentratie en veranderingen in genexpressie 6,7. De juistitiation en beëindiging van activerende signalen cruciaal passende T-celreacties. De activiteit van signaaleiwitten is afhankelijk van de vorming en ontbinding van eiwit-eiwit interacties posttranslationele modificaties zoals proteïne fosforylatie, vorming van eiwitcomplexen, eiwit ubiquitinering en de rekrutering van diverse cellulaire eiwitten plaatsen 8. Inzicht in de werking van de T-cel activatie proces is voor zowel immunologisch onderzoek en klinische toepassingen. Verschillende assays werden ontwikkeld om eiwit-eiwit interacties te onderzoeken, maar niet biochemische assays, zoals de gebruikte co-immunoprecipitatie werkwijze niet toe eiwit locatie te onderscheiden, waardoor elke waarneming waardevolle inzichten in de dynamiek van cellulaire mechanismen. Bovendien, deze massa assays combineert gewoonlijk eiwitten uit verschillende cellen die mogelijk in verschillende stadia van de tHij onderzocht cellulaire proces. Dit kan een nadelig effect op temporele resolutie hebben. Het gebruik van real-time beeldvorming van levende cellen kunnen zowel de ruimtelijke volgen van eiwitten en het vermogen om tijdelijk onderscheiden signalerende gebeurtenissen, dus licht werpen op de dynamiek van het proces 9,10. We stellen een werkwijze real-time imaging van signaal-complexvorming in T-cel activatie. Primaire T-cellen of T-cellijnen, zoals Jurkat, getransfecteerd met plasmiden die coderen voor eiwitten van belang gefuseerd aan monomere fluorescerende eiwitten, dat niet-fysiologische oligomerisatie 11. Levende T-cellen worden neergezet op een dekglaasje vooraf bekleed met T-cel activerende antilichaam 8,9, dat bindt aan het CD3/TCR complex, induceren T-celactivering terwijl overwinnen dat specifieke activerende antigenen. Geactiveerde cellen voortdurend afgebeeld met het gebruik van confocale microscopie. Beeldgegevens worden geanalyseerd om kwantitatieve resultaten, zoals col opbrengstocalization coëfficiënt van de signalering eiwitten.

We geven een voorbeeld van levende T-cell imaging en analyse. Voorafgaand aan de beeldvormende experiment werden SLP76 deficiënte T-cellen (J14) geanalyseerd met gebruik van FACS om de expressie van de fluorescerende eiwitten (Figuur 1) te bepalen. T-cellen getransfecteerd met de gelabelde signaaleiwitten mCFP-Nck en SLP76-mYFP werden afgezet over activeren slides en afgebeeld in T-cell spreiding (figuur 2). Verzamelde beelden tonen de dynamiek van eiwit lokalisatie in activering en ve...

Watch this Scientific Journal Video about Real-time Live Imaging of T-cell Signaling Complex Formation at JoVE.com

Real-time Live Imaging of T-cell Signaling Complex Formation

We beschrijven een live-cell imaging methode die inzicht geeft in eiwitdynamica tijdens de T-cel activatie proces. We tonen de totale gebruiksduur van de T-cel verspreiding assay, confocale microscopie en beeldanalyse te geven kwantitatieve resultaten volgen signalering complexvorming in T-cel activatie.

Real-time Live beeldvorming van T-cel signaleringscomplex Vorming

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Real-time beeldvorming van leukotriënen B 4 Mediated Cel Migratie en BLT1 Interacties met β-arrestine

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Een TIRF Microscopie Techniek voor Real-time, gelijktijdige beeldvorming van de TCR en de bijbehorende signaaleiwitten

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Real-time beeldvorming van heterotypische Bloedplaatjes-neutrofiele Interacties op het geactiveerd endotheel tijdens vasculaire ontsteking en trombose in Live Muizen

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Real-time beeldvorming van endotheelcellen-cel Junctions Tijdens Neutrophil Transmigration onder fysiologische Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Real-time beeldvorming van myeloïde cellen Dynamiek in<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinale Tumoren door draaiende schijf confocale microscopie

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Gelijktijdige Kwantificering van de T-cel receptor Excisie cirkels (TRECS) en K-verwijderen recombinatie Excisie cirkels (KRECs) van Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Beeldvorming van neutrofielen en monocyten in Mesenteriale Veins door Intravitale Microscopie op verdoofd muizen in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Beeldvorming CD4 T Cell interstitiële Migratie in de ontstoken Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Meting van de T-cel alloreactiviteit Met behulp van Imaging flowcytometrie

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Real-time Imaging en kwantificering van schimmel Biofilm ontwikkeling met behulp van een bewerking in twee fasen recirculatie-stroomsysteem

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...