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/pubmed?term=((MHC+and+T+cell+development%5BTitle%5D)+AND+%22Rev.+Immunogenet%22%5BJournal%5D)">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/pubmed?term=((Leukocyte+antimicrobial+peptides%3A+multifunctional+effector+molecules+of+innate+immunity%5BTitle%5D)+AND+%22J.+Leukoc.+Biol%22%5BJournal%5D)">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/pubmed?term=((Cytotoxic+T+cell+effector+and+memory+function+in+viral+immunity%5BTitle%5D)+AND+%22Curr.+Top+Microbiol.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Immune+responses+to+tumour+antigens%3A+implications+for+antigen+specific+immunotherapy+of+cancer%5BTitle%5D)+AND+%22J.+Clin.+Pathol%22%5BJournal%5D)">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/pubmed?term=((T-cell+antigen+receptor+genes+and+T-cell+recognition%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((The+actin+cytoskeleton+in+T+cell+activation%5BTitle%5D)+AND+%22Annu.+Rev.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Multiple+pathways+leading+from+the+T-cell+antigen+receptor+to+the+actin+cytoskeleton+network%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">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/pubmed?term=((Dynamic+molecular+interactions+linking+the+T+cell+antigen+receptor+to+the+actin+cytoskeleton%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading%3A+a+role+for+the+signal+transduction+adaptor+LAT%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">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/pubmed?term=((Imaging+techniques+for+assaying+lymphocyte+activation+in+action%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Quantifying+colocalization+by+correlation%3A+the+Pearson+correlation+coefficient+is+superior+to+the+Mander%27s+overlap+coefficient%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">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/pubmed?term=((Automatic+and+quantitative+measurement+of+protein-protein+colocalization+in+live+cells%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">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/pubmed?term=((Tyrosines+113%2C+128%2C+and+145+of+SLP-76+are+required+for+optimal+augmentation+of+NFAT+promoter+activity%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">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., Faragó, A., Downward, J., Buday, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+Nck+with+tyrosine-phosphorylated+SLP-76+in+activated+T+lymphocytes%5BTitle%5D)+AND+%22Eur.+J.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Functional+cooperation+between+the+proteins+Nck+and+ADAP+is+fundamental+for+actin+reorganization%5BTitle%5D)+AND+%22Mol.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Cooperative+interactions+at+the+SLP-76+complex+are+critical+for+actin+polymerization%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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. Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color. 5, rs3(2012).</li>
</ol>

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 observed spatially and over time, facilitating the monitoring of physiological processes as they occur and the detection of the cellular distribution of proteins. Various tools for the quantitative analysis of colocalization have been developed; Pearson's correlation coefficient remains a very attractive and widely available method for quantification of the degree of colocalization between two proteins 12. While fluorescence resonance energy transfer (FRET) experiments enable the detection of inter-protein interactions characterized by close proximity (10 nm), this method requires specialized settings (equipment and compatibility of fluorescent taggings used) and complex data processing. For most uses, colocalization analysis, in conjugation with biochemical methods, constitutes an easy-to-use and straightforward method for observing protein-protein interactions over time.
Establishing a fluorescence-intensity threshold for analyzed pixels, while ignoring pixels below the intensity threshold for either channel, is of critical importance for accurate and sensitive colocalization analysis. The commonly used algorithm for the objective and automated determination of these thresholds was developed by Costes et al. 13 and is implemented by image processing programs such as Imaris and Volocity.
Here, we demonstrate the use of the live T-cell imaging method for analyzing the dynamics of SLP76, a key scaffold in T cells, and Nck, an adaptor protein, both of which are essential for T-cell activation 14,15. Images of T cells collected during activation suggest that the two proteins are colocalized throughout the cellular activation process (Figure 3). This is quantitatively corroborated by performing the Pearson's colocalization test on images collected throughout the activation process. The Pearson's colocalization coefficients range from -1 to 1, where a score of -1 means anti-correlation, a score of 1 signifies perfect colocalization and 0 means no correlation between the two image channels. Comparison of Pearson's colocalization coefficients calculated for different time points and for different cells indicates that colocalization of Nck and SLP76 does not significantly change during cellular activation, but remains constant (p&gt; 0.3, Figure 4). While the overall colocalization coefficient of these proteins does not change throughout the activation process, it does not eliminate the possibility of a dynamic process of association and disassociation occurring between these proteins 18.
The protocol we presented can also be adapted to other hematopoietic cell lines by adjusting cell-growth conditions as required, selecting a transfection setting suitable for the cell line in question and modifying the cellular activation process, if applicable. Please note that while we used the LSM 510 Meta confocal microscope, with the appropriate adjustments and use of settings, other confocal microscopy systems can be successfully used as well.
The use of live T-cell imaging allows the regulatory and activation processes to be directly monitored, enabling signaling events to be explored with temporal and spatial resolution unavailable using biochemical and molecular methods. We present a straightforward method for carrying out a live T-cell spreading assay, monitoring relevant proteins in real-time and analyzing the data to yield quantitative results. Resulting microscopy data can also be used for other downstream applications, such as cell-shape index analysis 16 and FRET analysis 17,18, depending on the specific experimental setup and research objectives.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td></tr><tr><td>Amaxa human T Cell nucleofector kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Anti CD3 (UCHT1 clone) </td><td>BioLegend</td><td>300432</td><td></td></tr><tr><td>Falcon FACS Tubes</td><td>Becton Dickinson</td><td>352058</td><td></td></tr><tr><td>FCS</td><td>HyClone</td><td>SV30160.03</td><td></td></tr><tr><td>G418</td><td>Calbiochem</td><td>345810</td><td></td></tr><tr><td>German coverglass system 4 chamber slides </td><td>Lab-Tek II</td><td>155382</td><td></td></tr><tr><td>HCl</td><td>Bio Lab</td><td>8410501</td><td></td></tr><tr><td>Hepes </td><td>Biological Industries</td><td>03-025-1B</td><td></td></tr><tr><td>Hygomycin</td><td>Enzo</td><td>ALX-380-306</td><td></td></tr><tr><td>L-glutamine </td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>PBS (10X)</td><td>Sigma</td><td>D1408</td><td></td></tr><tr><td>Penicillin-Streptomycin </td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>Poly-L-lysine 0.1 % (w/v) in H2O</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>RPMI</td><td>Sigma</td><td>R87589</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </td></tr><tr><td>Accublock digital dry bath</td><td>Labnet</td><td>D1105A</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Centrifuge 5810 R</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 510 Meta</td><td></td></tr><tr><td>Heatable mounting frame - Heating Insert P S </td><td>PeCon</td><td>130-800-031</td><td></td></tr><tr><td>TempModule S (required for the heatable mounting frame)</td><td>Zeiss</td><td>411860-9010-000</td><td></td></tr><tr><td>Electroporation device</td><td>Amaxa</td><td>Nucleofector I</td><td></td></tr><tr><td>Fluorescence activated cell sorter</td><td>Becton Dickinson</td><td>FACSVantage SE</td><td></td></tr><tr><td>Image analysis software</td><td>Bitplane</td><td>7.0.0</td><td></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 (Figure 3). Computerized image processing provides new insights and quantitative information from the visualized cells, while minimizing human bias. To examine the colocalization of the two proteins throughout the cellular activation process, we utilized the colocalization tool of the Imaris software (Bitplane AG, Zurich Switzerland). By comparing the colocalization coefficients between different time points across imaged cells, we were able to ascertain that the colocalization of Nck and SLP76 does not change significantly throughout T-cell activation (p &gt; 0.3) (Figure 4).
<img alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50076/50076fig1highres.jpg" src="/files/ftp_upload/50076/50076fig1.jpg" /> 
Figure 1. FACS analysis of cells double transfected with fluorescently-labeled proteins. Data are shown as fluorescence intensity histograms for (A) mCFP; (B) mYFP; and (C) as a dot plot. Fluorescence intensity of J14 cells transfected with mCFP-Nck and SLP76-mYFP are represented in cyan and yellow, respectively. Untransfected J14 negative control cells are indicated by black lines.
<img alt="Figure 2" fo:content-width="4in" fo:src="/files/ftp_upload/50076/50076fig2highres.jpg" src="/files/ftp_upload/50076/50076fig2.jpg" /> 
Figure 2. A schematic representation of the live T-cell spreading assay. (A) The preparation of T-cell activating slides; (B) live T-cell spreading assay and analysis; (C) microscopy setup. <a href="https://www.jove.com/files/ftp_upload/50076/50076fig2large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50076/50076fig3highres.jpg" src="/files/ftp_upload/50076/50076fig3.jpg" /> 
Figure 3. Distribution of Nck and SLP76 during the T-cell activation process. Cells expressing mCFP-Nck and SLP76-mYFP were dropped over anti-CD3 stimulatory antibody pre-coated coverslips and constantly imaged for a period of 7 min. mCFP-Nck is represented by cyan, while SLP76-mYFP is shown in yellow.
<img alt="Figure 4" fo:content-width="4.4in" fo:src="/files/ftp_upload/50076/50076fig4highres.jpg" src="/files/ftp_upload/50076/50076fig4.jpg" /> 
Figure 4. Quantitative analysis of protein colocalization. Image processing of cells was performed using the Imaris software (Bitplane AG, Zurich Switzerland). Colocalization is calculated as Pearson's correlation coefficient between the intensities of the mCFP channel and the mYFP channel for each pixel. Pearson's colocalization coefficients calculated from different time points are compared. Thirty seconds into the activation process, the colocalization coefficient of mCFP-Nck and SLP76-mYFP was increased significantly (p&lt;0.05). These results were calculated by analyzing &gt;50 independent cells. Mean &plusmn; standard error are shown.

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 | 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 ...

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

Nanyang Technological University

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

Cellular Processes

Cell Signaling

SCIENTISTS IN ACTION

13.8K 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

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 ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Biochemistry

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 ...

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 ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Imaging the Human Immunological Synapse

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

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

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

High-Resolution Imaging Technique for Detecting Ca2+ Microdomains in T Cells

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Source: Steblyanko, M., et al. <a href="https://app.jove.com/v/58143">Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of CD4+ T Cells for an ...