The authors thank Sophia Fried for technical assistance. MBS thanks the following agencies for their research support: The Israel Science Foundation for grants no.1659/08, 971/08, 1503/08 and 491/10, the Ministries of Health &amp; Science for grants no. 3-4114 and 3-6540, the Israel Cancer Association through the Estate of the late Alexander Smidoda, and the Taubenblatt Family Foundation for the Bio-medicine excellence grant.

1. T-cell Transfection
<ol>
	<li>Prepare the activated Amaxa Nucleofector solution by mixing the kit's "Supplement" solution to the Nucleofector Solution. The activated solution may be stored at 4 &deg;C for up to three months.</li>
	<li>Grow Jurkat T cells in culture medium [10% fetal calf serum (FCS), 1% Penicillin-Streptomycin solution, and 2 mM L-glutamine in RPMI]. Optimally, cells should be used 1-2 weeks after thawing. Using cell cultures in logarithmic growth is crucial for high transfection efficiency and cell survival. Alternatively, with minor modifications, this protocol can be used with primary T cells, as previously described 8. It should be emphasized that as the duration of the activation process of primary T cells is longer than that of Jurkat T cells (~30 min), a microscope stage mounted incubation system must be used to maintain CO2, humidity levels and temperature throughout the activation process.</li>
	<li>Collect 5 &times; 106 log-phase T cells per transfection into a 15-ml tube.</li>
	<li>Centrifuge cells for 7 min at 400 &times; g at room temperature.</li>
	<li>During centrifugation, prepare a 6-well plate. Fill one well for each transfection with 5 ml culture medium, and pre-incubate at 37 &deg;C.</li>
	<li>Collect the tubes from the centrifuge and discard the supernatant. Resuspend the cell pellet in 1 ml RPMI.</li>
	<li>Centrifuge cells for 7 min at 400 &times; g at room temperature.</li>
	<li>During centrifugation, prepare the transfection solution. In one well of a 96-well plate, mix activated 100 &mu;l Amaxa Nucleofector solution prepared in step 1 with 5 &mu;g DNA of the plasmid of interest encoding a fluorescently tagged protein. Optimally, DNA concentration should be greater than 1 &mu;g/ml in order to avoid diluting the transfection solution.</li>
	<li>Mix well by gentle pipetting.</li>
	<li>After centrifugation of the cells, carefully remove all the supernatant while not disturbing the pellet.</li>
	<li>Pipette the DNA/transfection solution twice.</li>
	<li>Add the DNA/transfection solution to the pellet.</li>
	<li>Transfer the cells to the Amaxa cuvette.</li>
	<li>Insert the cuvette into the Amaxa Nucleofector device.</li>
	<li>Use the H-10 Amaxa transfection setting for electroporation (for Jurkat ) or T-23 (for primary T cells).</li>
	<li>Remove the 6-well plate from the incubator.</li>
	<li>Following electroporation, carefully transfer the supernatant with the cell suspension to the 6-well plate using a Pasteur pipette. A pellet consisting of cell debris may form. Avoid transferring the pellet.</li>
	<li>Incubate the cells for 24 hr at 37 &deg;C.</li>
	<li>Transfer 2.5 ml of cell suspension from each well into a new well. Add 2.5 ml warm culture medium (described in step 2) to each well.</li>
	<li>After 72 hr, transfer the cells to a sterile tube and centrifuge for 7 min at 400 &times; g at room temperature. Discard the supernatant and replace it with culture medium supplemented with the mammalian cell selection antibiotic appropriate for the plasmid used (apply a concentration suitable for the antibiotics used; we used G418 and/or Hygromycin in concentrations of 1.3 mg/ml and/or 0.2 mg/ml, respectively). Culture the cell suspension in a fresh well. Alternatively, cells can be transfected transiently and imaged 48 hr after transfection. To prepare transiently transfected cells, co-transfect the cells with the two plasmids simultaneously at step 8, continuing normally to step 20, and then skip directly to step 23. Note that under this scenario, the cells should be used immediately, and generally their fluorescence intensity is heterogeneous and not necessarily high.</li>
	<li>Grow cells with culture medium supplemented with the appropriate selection antibiotic. Split the cells and supplement them with antibiotic-supplemented culture medium as needed.</li>
	<li>After 2 weeks, verify successful transfection with the use of FACS (fluorescence-activated cell sorting) as follows:</li>
	<li>Collect 106 transfected cells, 106 mock transfected cells (negative control), and 106 cells that are known to stably express the fluorescently tagged protein. If cells are transfected with more than one fluorescent protein (see step 27), use multiple, singly tagged cell lines as positive controls.</li>
	<li>Centrifuge cells for 7 min at 400 &times; g at room temperature.</li>
	<li>Discard the supernatant. Resuspend cells in 500 &mu;l FACS buffer (PBS w/o Ca2+ and Mg2+, 5% FCS, 0.05% sodium azide).</li>
	<li>Use an analytical FACS to verify cellular fluorescence. Compare fluorescence of the transfected cells to that of the negative and positive controls.</li>
	<li>To transfect the cells with an additional, fluorescently tagged protein, repeat steps 3-26 using the transfected cells.</li>
	<li>Cells displaying high levels of fluorescence should be used. Optimally, at least 50% of the cells in the culture should be highly fluorescent. Cell fluorescence may be increased by cell sorting via FACS.</li>
</ol>
Any additional tagged proteins must be fused to different fluorophores and be encoded on plasmids encoding for different selection antibiotics. Selection medium for cells transfected with more than one plasmid should be supplemented with all the appropriate antibiotics.
2. T-cell Spreading Assay Preparation
<ol>
	<li>Use Lab-Tek II German coverglass system 4 chamber slides. Make sure not to accidently scratch the inner surface of the chambers, e.g. with a pipette tip, during preparation or use.</li>
	<li>Prepare 50-ml slide preparation solution: Take 4.6 ml of 12 M (37%) HCl, add 38.5 ml 100% ethanol, and 6.9 ml doubly distilled water.</li>
	<li>Fill each chamber of the slide with 500 &mu;l slide preparation solution.</li>
	<li>Incubate at room temperature for 10 min.</li>
	<li>Aspirate the chambers, drying them completely.</li>
	<li>Incubate the uncovered slides for 1 hr at 45 &deg;C till fully dry.</li>
	<li>Prepare 0.01% poly-L-lysine solution by diluting 0.1% poly-L-lysine (Sigma-Aldrich) in double distilled water.</li>
	<li>Apply 500 &mu;l to each chamber.</li>
	<li>Incubate the slide for 15 min at room temperature.</li>
	<li>Aspirate the chambers, drying them completely.</li>
	<li>Incubate for 3 hr at 45 &deg;C till fully dry.</li>
	<li>Coated slides can be stored dry for several months at room temperature.</li>
	<li>Prepare an activating antibody solution. Use an anti-CD3 antibody: either HIT3a (BD Pharmingen, #555337) or UCHT1 (BD Pharmingen, #555330), 10 &mu;g/ml in 400 &mu;l PBS, per chamber. This solution should be prepared shortly before slide preparation.</li>
	<li>Fill each chamber with 400 &mu;l of the activating antibody solution.</li>
	<li>Incubate the slide for 2 hr at 37 &deg;C or, preferably, overnight at 4 &deg;C.</li>
	<li>Remove the activating antibody solution and immediately wash the chamber twice with 300 &mu;l PBS. From this point, make sure the chambers remain filled with buffer.</li>
	<li>Store the PBS-filled slide at 4 &deg;C. We suggest using the prepared slide chambers within 1 week.</li>
</ol>
3. T-cell Activation and Imaging
<ol>
	<li>Prepare imaging buffer [1.25 ml HEPES (1 M), 10 ml FCS, 38.75 ml RPMI without phenol red]. Imaging buffer can be stored at 4 &deg;C after filtration.</li>
	<li>Activate the Zeiss LSM 510 Meta microscope. Warm the microscope heatable mounting frame to 37 &deg;C. Select "Expert mode".</li>
	<li>Click the "Acquire" tab and open the following tabs: Lasers; Microscopy; Configuration; Scan; Time Series.</li>
	<li>Activate the needed lasers. For excitation of mCFP and mYFP, set the Argon/2 laser to "standby" for 5 min, then to "on".</li>
	<li>If you have performed this live-cell imaging assay before, using the same fluorophores, open the resulting LSM file, click the "reuse" icon, and continue from step 9. Otherwise continue from step 6.</li>
	<li>Define channels using filters appropriate for the excitation and emission of the fluorescent proteins used.</li>
	<li>In the "Scan control" window, select the Z stack option.</li>
	<li>Enter the following scan parameters: Number of slices: 5; Interval: 0.5 &mu;m; Current slice: 3.</li>
	<li>Prepare an Accublock Digital Dry Bath (Labnet) at 37 &deg;C near the microscope.</li>
	<li>Warm the imaging buffer to 37 &deg;C.</li>
	<li>Prepare the stored slide by aspirating the PBS, replacing it with 600 &mu;l of warm imaging buffer.</li>
	<li>Incubate the slide at 37 &deg;C. Keep the slide in the incubator until step 13, for at least 15 min.</li>
	<li>Collect 5 &times; 105 transfected T cells.</li>
	<li>Centrifuge cells for 2 min at 400 &times; g.</li>
	<li>Discard the supernatant.</li>
	<li>Resuspend cells in 1 ml warm imaging buffer, and transfer them to a 1.5-ml Eppendorf tube.</li>
	<li>Place the test tube on the Accublock Digital Dry Bath.</li>
	<li>Take the warmed slide from the incubator, and mount it on the warmed microscope mounting frame.</li>
	<li>Click the "VIS" icon.</li>
	<li>Mix the cell suspension and remove 1-2 &mu;l.</li>
	<li>Insert the tip of the pipette into the imaging buffer above the bottom of the slide. Avoid scratching the slide.</li>
	<li>Seed the cells over the objective's aperture.</li>
	<li>Immediately begin visually tracking the fluorescent cells as they descend. Select a cell that displays high fluorescence in both channels.</li>
	<li>Before the cell reaches the surface of the slide, activate the LSM mode.</li>
	<li>Toggle only one excitation laser. Select a laser for which you have a DIC channel.</li>
	<li>Set Zoom to 1. Click the "Fast XY" icon. Click the "Stop" icon. Using the Crop tool, select an ROI centered on the cell.</li>
	<li>Set Zoom to 3. Click the "Fast XY" icon, then manually set the focus to optimally view the cell. Click the "Stop" icon.</li>
	<li>Toggle all the needed excitation lasers.</li>
	<li>Begin recording by clicking the "StartT" icon. Image the cell for the entirety of the spreading process (commonly 5 min). When finished, click the "Stop" icon.</li>
	<li>Save the file.</li>
	<li>To acquire additional cells, click the "VIS" icon. If there are cells that did not yet come into contact with the slide's bottom, continue from step 23. Avoid imaging cells that have already begun the spreading process. Otherwise proceed to step 32.</li>
	<li>If the observed slide area is mostly devoid of cells, add another aliquot of cells (continue from step 20). Otherwise proceed to step 33.</li>
	<li>To acquire additional cells, move the slide so that the area above the objective's aperture is devoid of cells.</li>
	<li>Add another aliquot of cells (and continue from step 20).</li>
	<li>After performing all the desired imaging, open a saved LSM file.</li>
	<li>Click "Gallery", "Time+Z" and "Subset".</li>
	<li>Select the relevant time range and Z stack.</li>
	<li>Save the new file.</li>
	<li>Perform steps 35-38 for all the imaging files.</li>
</ol>
4. Imaging Data Analysis
<ol>
	<li>Open the LSM files using Imaris software (Bitplane AG, Zurich Switzerland).</li>
	<li>Click "Surpass". Click the "Image Processing" menu, and select "Swap Time and Z". This will assist in the correct cropping of the image.</li>
	<li>Click the "Edit" tool bar and select the "Crop 3D" option in the drop menu. Crop the image to include only the area surrounding the cell. We suggest cropping a 300 &times; 300 pixel square centered on the cell. Note that the shape and location of the cell can change over time. Observing the t axis, make sure that the cell is within the cropped region at all time points.</li>
	<li>Click the "Image Processing" menu, and select "Swap Time and Z" to restore the original temporal separation of the image.</li>
	<li>Press the "Coloc" button on the main tool bar. Select "Build Time Dep. Coloc". Imaris will automatically calculate intensity thresholds for each channel at each time point.</li>
	<li>View the results of the colocalization analysis by selecting "Channel Statistics". Export the data by clicking "Export".</li>
	<li>Repeat steps 1 to 6 for each imaged cell. We recommend that at least 50 cells be analyzed in this manner.</li>
	<li>Calculate the mean Pearson's colocalization coefficient and its standard error or deviation.</li>
</ol>

<ol>
	<li>Viret, C., Janeway, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+and+T+cell+development.">MHC and T cell development.</a> Rev. Immunogenet. 1, 91-104 (1999).</li><li>Risso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+antimicrobial+peptides:+multifunctional+effector+molecules+of+innate+immunity.">Leukocyte antimicrobial peptides: multifunctional effector molecules of innate immunity.</a> J. Leukoc. Biol. 68, 785-792 (2000).</li><li>Doherty, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+cell+effector+and+memory+function+in+viral+immunity.">Cytotoxic T cell effector and memory function in viral immunity.</a> Curr. Top Microbiol. 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. H. <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+cytoskeleton+in+T+cell+activation.">The actin cytoskeleton in T cell activation.</a> Annu. Rev. 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. 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We have no conflicts of interest to disclose.

The regulation and function of multiple cellular processes depend on the formation and termination of protein-protein interactions. Microscopic imaging allows the real-time tracking of fluorescently tagged proteins in living cells. Colocalization of tagged proteins can suggest a direct or indirect interaction between the proteins, and can be used to reinforce findings obtained by biochemical methods, such as immunoprecipitation. Unlike biochemical methods, live-cell imaging allows these inter-protein interactions to be o...

<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

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We present an example of live T-cell imaging and analysis. Prior to the imaging experiment, SLP76 deficient T cells (J14) were analyzed with the use of FACS to determine the expression of the fluorescent proteins (Figure 1). T cells transfected with the tagged signaling proteins mCFP-Nck and SLP76-mYFP were deposited over activating slides and imaged during T-cell spreading (Figure 2). Collected images show the dynamics of protein localization throughout activation and spreading (<strong...

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Real-time Live Imaging of T-cell Signaling Complex Formation | Immunology and Infection | Video

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

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

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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13.6K Views.  Bar-Ilan University. We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. ...