We acknowledge all the past and present members of the lab for their generous contribution. This work was supported by grants from the Spanish Ministerio de Econom&#237;a y Competitividad (MINECO), Plan Nacional de Investigaci&#243;n Cient&#237;fica (SAF2016-77561-R to M.I., which was in part granted with FEDER-EC funding). We acknowledge Facultad de Medicina (UAM) and Departamento de Audiovisuales of the Facultad de Medicina for their support and the facilities provided to produce the video. We acknowledge NIKON-Europe for the continuous and superb technical and theoretical support. Free access to this article is sponsored by Nikon.

1. Preparation of Slides to Adhere Raji Cells
<ol>
	<li>Add 150 &#181;L of fibronectin (100 &#181;g/mL) per well to an 8 microwell chamber slide (plastic-bottom chamber slide) and incubate it for 30 min to 1 h at 37 &#176;C. This adhesion substrate will allow the binding of Raji cells to the well bottom (Step 2), the formation of living conjugates with Jurkat cells (Step 4) cells, and time-lapse microscopy capture (step 6), and is also compatible afterwards with optional paraformaldehyde (PFA) fixation (step 7). 
	NOTE: For acetone fixation, use glass bottom chamber slides and poly-L-lysine (20 &#181;g/mL) instead of fibronectin, since acetone dissolve plastic. The 8 microwell chamber slide (1 cm2 well, 300 &#181;L maximum volume) or equivalent is a flexible and appropriate format.</li>
	<li>Aspirate fibronectin using a 200 &#956;L automatic pipette and wash each well with 200 &#181;L of PBS for 2 min with gentle shaking. Repeat this wash one more time. The chamber slide can be stored at this stage with PBS for 1-2 weeks at 4 &#176;C.</li>
</ol>
2. Adhesion of Raji Cells to the Chamber Slides and 7-amino-4-chloromethylcoumarin (CMAC) Labeling
<ol>
	<li>Transfer 10 mL of a confluent (1-2 x 106 cells/mL) pre-culture of Raji cells to a 15 mL, V-bottom tube. Mix well and use 10 &#181;L to count the cells on Neubauer chamber or equivalent.</li>
	<li>Centrifuge the remaining cells at 300 x g for 5 min at room temperature. Aspirate and discard the supernatant.</li>
	<li>Gently resuspend the cell pellet in warm complete culture medium (RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin) at a concentration of 106 cells/mL. Use the equation given below: 
	([ ]initial x Vinitial = [ ]final x Vfinal), where [ ]initial represents initial cell concentration, Vinitial = initial volume of the cell suspension, [ ]final = final cell concentration, Vfinal = final volume of the cell suspension. 
	NOTE: Depending on the cell concentration of the starting culture, it is possible to collect more cells than needed but it is important to maintain the remaining cells in culture (37 &#176;C) till the end of the experiment in order to prevent potential problems (see Step 2.6).</li>
	<li>Label Raji cells to allow for their identification during the synaptic conjugate formation. In this experiment, 7-amino-4-chloromethylcoumarin (CMAC) labeling is performed in step 2.4.2.
	<ol>
		<li>Transfer the required number of Raji cells in the culture medium to a 2 mL tube. For the 8 microwell chamber slide, a total of 1.6 mL of the cell suspension is needed (200 &#181;L per well).</li>
		<li>Add CMAC to a final concentration of 10 &#181;M. Keep the cells in the dark by covering the tube with aluminum foil since CMAC is light-sensitive. 200 &#181;L containing 2 x 105 Raji cells are needed per 1 cm2 well. Thus, if 8 wells need to be prepared, 1.6 x 106 Raji cells are required. 
		NOTE: Labeling Raji cells with cell tracker blue (CMAC, UV excitation and blue emission) distinguishes them from Th cells when the synaptic conjugates are formed. This dye is compatible with PFA and acetone fixatives and allows further immunofluorescence procedures. Try to avoid light exposition. The labeling of Raji cells in a pool with CMAC followed by resuspension before the adhesion of the Raji cells to the fibronectin-coated chamber slides ensures the homogeneous labeling of Raji cells with CMAC among different wells.</li>
	</ol>
	</li>
	<li>Resuspend CMAC-stained cells and, after aspiration of the PBS in the chamber slide from step 1.2., transfer 200 &#181;L of the cell suspension to each well of the fibronectin-coated chamber slides prepared in step 1.1-1.2. Incubate the chamber slide at 37 &#176;C, 5% CO2 for 30 min-1 h. 
	NOTE: The adhesion and CMAC labeling will simultaneously occur at this step and this saves time. Please be aware that Raji cells will sediment quickly and caution needs to be taken to maintain a homogeneous concentration in the cell suspension before seeding. It is not necessary to wash CMAC at this stage by centrifugation, since CMAC washing will be more easily done at step 2.7 (when the labeled Raji cells are already adhered to the chamber slides). Since CMAC is present in the cell suspension in large excess, the blue fluorescence background is too high to distinguish the blue-stained cells. Check cell CMAC fluorescence in step 2.7 after CMAC washing.</li>
	<li>Ensure that Raji cells are adhered to the bottom of the wells by gentle shaking of the chamber slides on the microscope. Ensure that the cells display gaps among each other and are not confluent (Figure 1, middle panel). 50-60% of cell confluence is appropriate.
	<ol>
		<li>If most of the cells efficiently adhere to the chamber slide and no cell gaps are observed, wash each well with warm complete medium and resuspend the medium with a 200 &#956;L automatic pipet to detach cell excess. Check confluence after each resuspension step.</li>
		<li>If the cells do not adhere, repeat the adhesion step again and increase adhesion time and/or cell number. 
		NOTE: It is possible to stop here, incubate the chamber slide at 37 &#176;C, 5% CO2 overnight (O/N), and continue with the protocol the next day. Please confirm the next day that Raji cells remain adhered and CMAC-labeled by using fluorescence microscopy.</li>
	</ol>
	</li>
	<li>Wash each well again carefully with warm supplemented RPMI to eliminate excess CMAC and check for blue emission with the fluorescence microscope (Figure 1). 
	NOTE: To avoid the use of immersion oil (sticky and viscous) and high numerical aperture oil objectives, extra-long distance (i.e., 20x or 40x) objectives can be used to quickly check CMAC labeling using the fluorescence microscope.</li>
</ol>
3. Pulse of CMAC &#8212; Labeled Raji Cells with Staphylococcal Enterotoxin E
<ol>
	<li>Add Staphylococcal Enterotoxin E (SEE, 1 &#181;g/mL) to each well. SEE can be conveniently diluted in cell culture medium (working solution at 100 &#181;g/mL) from the SEE frozen stocks (1 mg/mL in PBS). Use 2 &#956;L of the 100x working solution per 200 &#956;L microwell. 
	CAUTION: Use gloves for this step and dispose of the used tip into the biohazard box.</li>
	<li>Incubate the chamber slide at 37 &#176;C, 5% CO2 for at least 30 min. The SEE effect lasts for at least 3-4 h. 
	NOTE: SEE can be added to the wells at different time points when required, if distinct time-lapse setups are planned (step 5) and/or depending of the start time point for the end point experiments (step 6).</li>
</ol>
4. Preparation of Jurkat Cells
<ol>
	<li>Use a previously growing culture of Jurkat cells (1-2 x 106 cells/mL) for this experiment. Use cells from a standard culture flask or from a previous transfection following standard electroporation protocols, as previously described23. The transfection of Jurkat cells will allow time lapse visualization of the traffic of secretory granules in living cells. For instance, when GFP-CD63 (a marker of MVB) is expressed the movement of GFP-CD63-decorated vesicles can be recorded (Video 1).</li>
	<li>Observe the cells under a phase contrast microscope. If an excess of dead cells (&#62;20-30%) are observed, perform Ficoll density gradient centrifugation using standard protocols24, to eliminate the excess of dead cells (dead cells exhibit higher density than living cells) prior to use (see Discussion).</li>
	<li>Transfer the cells to a 15 mL tube, V-bottom tube, and use 10 &#181;L for counting using hemocytometer.</li>
	<li>Centrifuge the remaining cells as described in step 2.2. Discard the supernatant and resuspend the cells at the same concentration as Raji cells (1 x 106/mL) using fresh, warm culture medium. Follow steps 2.2-2.3.</li>
	<li>Maintain the Jurkat cells in culture (37 &#176;C, 5% CO2) while waiting for the Step 4. 
	NOTE: In the second option (transfection), the number of living cells is going to be much lower than in the first one. Thus, consider using a higher starting cell culture volume in order to have enough cells for the experiment. From 10 x 106 Jurkat cells per electroporation cuvette and transfection, only 2-4 x 106 Jurkat cells will survive after 48 h of transfection and some of these cells will be lost during the Ficoll step. Thus, one electroporation cuvette is generally sufficient to challenge the adhered SEE-pulsed Raji cells from 8 micro wells (1.6 x 106 transfected Jurkat cells needed).</li>
</ol>
5. Co-seeding of Raji and Jurkat Cells
<ol>
	<li>Take the chamber slides containing the CMAC-labeled, SEE-pulsed, adhered Raji cells out of the incubator from step 3.2. It is not necessary to wash the CMAC at this stage since this was previously done in Step 2.7.</li>
	<li>Aspirate carefully the culture medium of each well, one by one, from one corner of the well using an automatic 200 &#956;L pipette. Do not let the medium in the well dry out completely.</li>
	<li>Immediately replace the medium with 200 &#181;L of resuspended Jurkat cells in cell culture medium (1 x 106/mL) prepared in step 4.5. If time lapse imaging is performed, go to step 6 immediately after this step, since Jurkat cells tend to sediment and form synaptic conjugates very quickly. For convenience, the microwells containing SEE-pulsed, adhered Raji cells that do not receive seeding with Jurkat cells at this stage should be maintained with cell culture medium until subsequent challenge with Jurkat cells. This will flexibly allow subsequent challenge with Jurkat cells for additional time lapse or reverse kinetic, end point experimental approaches.</li>
	<li>If a time lapse is going to be performed, quickly proceed to step 6. This involves the co-culture for 1-2 h on the microscope stage incubator or equivalent at 37 &#176;C, 5% CO2 to allow the synaptic conjugate formation and simultaneous image acquisition. If end point analysis, but no time lapse, is foreseen check conjugate formation after the co-culture period using the microscope (as in Figure 1) before fixing the cells (step 7).</li>
</ol>
6. Time Lapse Imaging of Emerging Synaptic Conjugates
<ol>
	<li>Prepare the microscope and incubation chamber prior to imaging. For the example shown in Video 1, the detailed microscopy settings are shown in Figure 2. 
	NOTE: If a time lapse experiment is planned, all the microscope settings and complements (ambient cell culture chamber, etc.) should be prepared before adding the Jurkat suspension to the chamber slide with adhered Raji cells. The following steps are described for a commercial microscope (Table of Materials). However, any inverted fluorescence microscope equipped with a cell culture incubator can be used.
	<ol>
		<li>Use a microscope with a 60x oil-immersion, high numerical aperture when imaging polarized traffic.</li>
		<li>Ensure that the automatic focus system is switched on and adjust the offset to focus the Raji cells bound to the bottom surface. Please refer to Figure 1, Video 1 and Video 2.</li>
	</ol>
	</li>
	<li>After Jurkat addition to each well containing the adhered Raji cells in step 5.3, quickly locate the microwell chamber slide on the pre-heated (1-2 h) microscope stage incubator (i.e., OKOlab) and select some XY positions with the microscope, fields in which it is likely to record an emerging IS formation made by, for instance, a Jurkat-transfected cell falling into the microscope focus.</li>
	<li>Use a pre-heated microscope stage incubator since it was observed that a temperature-stabilized stage maintains stable X,Y,Z positions. Criteria for a convenient XY field are: well-focused and non-confluent Raji cells (i.e., displaying gaps among cells) and the presence of transfected Jurkat cells (this can be checked by combining transmittance and UV or GFP channels). Jurkat cells will sediment very quickly (few minutes) on the chamber slide, and the chances to image emerging synapses will decrease with time (Figure 1). It is possible either to finish the experiment after the defined time lapse or proceed to the Step 7 and fix the conjugates for subsequent immunofluorescence and analyses. 
	NOTE: It is possible to select up to 16 different microscope fields from up to 4 different microwells for simultaneous, multi-well time-lapse acquisition with the proper temporal resolution (1-2 min per frame). The limitation relies on both the number and intensity (affecting camera exposition) of the diverse fluorochromes to be imaged (dependent on the number of expressed fluorescent proteins, apart from CMAC). One way to increase the frame rate is recording for the CMAC channel in only one out of each &#34;n&#34; time frames for GFP (i.e., n = 8, as shown in Figure 2), since Raji cells are adhered to the well bottom and do not easily move as Jurkat cells. In addition, this benefits the cell viability, since frequent UV light exposure may damage the cells. Try to adjust the time frame rate to 1 frame every 1 minute or less (i.e., 20 s per frame in Video 1, Figure 2) since the polarization of MVB takes a few min to hours to complete. A microscope equipped with a motorized epi-fluorescence turret and appropriate band-pass fluorescence filters or equivalents are necessary to perform this multichannel capture.</li>
</ol>
7. End Point Formation of Synaptic Conjugates and Fixation
<ol>
	<li>If only an endpoint experiment is planned (1-2 hours incubation to allow synaptic conjugate formation is appropriate), incubate the chamber slide at 37 &#176;C, 5% CO2 for 1-2 h. Check conjugate formation after the culture period (as in Figure 1) and, subsequently, fix the conjugates with acetone or PFA (fixation will depend on the antigens and antibodies to be used in the subsequent immunofluorescence). In this case the incubation does not require a microscope stage incubator. Please refer to Video 3 for an example.</li>
	<li>To fix the cells, wash the well by gentle shaking with warm RPMI (37 &#176;C) medium without FCS (albumin from serum may precipitate with acetone fixation). Aspirate and add 200 &#181;L of PFA or pre-chilled acetone to each well. Incubate the chamber slide at room temperature (RT) or on ice, respectively, for 20 min. 
	NOTE: For the acetone fixation, pre-chill the acetone at -20 &#176;C and pre-chill the chamber slide at 4 &#176;C. Remove plastic lids when acetone is used to fix the cells cultured in the 8 well glass-bottom chamber slides.</li>
	<li>Wash each well twice with PBS and add 200 &#181;L of quenching solution (PBS, 50 mM NH4Cl). Incubate the chamber slide at 4 &#176;C. 
	NOTE: At this stage the chamber slide can stay for at least one month at 4 &#176;C before performing the immunofluorescence protocol, as described8. The lid prevents evaporation.</li>
</ol>
8. Image Processing
<ol>
	<li>Perform post-acquisition image deconvolution (i.e. Huygens deconvolution or equivalent, Table of Materials) of time-lapse series and/or still photos of fixed cells. Deconvolute by employing an appropriate software (i.e., using the &#34;wide field&#34; optical option in Huygens) and the correct optical parameters. Deconvolution necessitates for the image processing of the measured point spread function (PSF) of the microscope4.</li>
	<li>Alternatively, use the software to calculate the idealized PSF by automatic loading of the optical parameters included among the metadata from the microscope files. These optical parameters comprise fluorochrome wavelength, refraction index, numerical aperture of the objective, and the imaging technique (confocal, wide-field, etc.)4.</li>
	<li>Subsequently, the imaging software uses the PSF and diverse deconvolution algorithms (i.e., QMLE and CMLE in Huygens software) in a step-by-step accumulative, calculation process, whose results than can be continuously visualized and stopped (or resumed) when required by the user. At this stage the user can change the number of convolutions and/or the signal to noise ratio and resume deconvolution. The deconvolution software works well with time lapse series (X,Y,T) (Video 2) and Z-stacks (X,Y,Z) (Video 3). The deconvoluted channels were subsequently merged to the CMAC, raw channel, since cytosolic, diffuse fluorochromes do not improve by deconvolution4,9.</li>
</ol>

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The authors declare no conflict of interest.

A limitation of this protocol is that not all synapses will be ideally oriented perpendicular to the optical axis. Using this technique, there is no way to predict and/or to influence the ideal orientation for immune synapse imaging. To fix this problem, we exclude from the subsequent analyses all the randomly captured synapses that finally do not fulfill the ideal criteria. These synapses, opportunely enough, are not very frequent. However, it is possible to circumvent this limitation using several experimental approaches4.
The polarized CD63 release (degranulation) can be quantified by other complementary techniques such as cell surface staining of CD63 (CD63 relocalization to the cell surface) in living cells (not fixed and not permeabilized), after step 6, and subsequent washing and fixation, as previously shown8. In addition, CD63 release on exosomes8,9 and exosome quantification by nanoparticle tracking analyses9,25 can be performed. These approaches are certainly compatible with our protocol, provided fixation is being performed after the cell surface immunofluorescence of living cells.
We have found the ideal number of cells (for a 1 cm2 well in the 8-well chamber slide) is 4 x 105 cells (2 x 105 Raji cells and 2 x 105 Jurkat cells) since they adhere efficiently to the bottom of the well. Binding efficiency to plastic (using fibronectin), or glass bottom (using poly-L-lysine) microslides is not usually a problem. Higher cell numbers may produce no gaps among adhered cells and, subsequently, complex synaptic conjugates (Figure 1); both situations are not desirable when single cell-to-cell conjugates need to be imaged in, for instance, MTOC or secretory granule polarization experiments. Lower number of cells may decrease the chances to find conjugates, especially when transfected Jurkat cells are challenged with APC to generate synapses. Please note that we have observed that a temperature-stabilized stage incubator in place on the microscope X/Y stage before the onset of the protocol (i.e., 1-2 h in advance to stabilize the stage/microscope setup) maintains stable X,Y,Z parameters, which is crucial for proper imaging. Automatic focus system will eventually compensate small Z variations.
When certain gene transduction techniques such as electroporation are used to express fluorescent chimeric proteins (i.e., GFP-CD63) in the Jurkat clones (Video 1), a considerable fraction of cells may die after electroporation. This can be a problem since although dead cells do not form synapses, when they are in excess over the living, transfected cells, they may interfere with the formation of conjugates made by living Th cells. We have found that careful elimination of dead cells from transfected cultures by using a density gradient medium following standard protocols before the conjugate formation step can indeed increase the chances to properly image conjugates. In addition, low transfection efficiencies (&#60;20%) can be an important caveat since this, combined with a moderate conjugate formation efficiency (around 60%)25, will decrease the probability to find conjugates made by transfected cells. This is not a problem when non-transfected cells are used to obtain conjugates in end point experiments and subsequent fixation. The 8 microwell chamber slides are compatible with conventional immunofluorescence protocols. This increases the flexibility of the above protocol with different purposes. Fixation with acetone can be a problem to consider when using chamber slides with plastic-bottom wells. However, there are commercially available 8 microwell microscope chamber slides containing glass bottoms, which are compatible with acetone fixation. Remove plastic lids when acetone is used to fix the cells cultured in 8 microwell glass-bottom chamber slides.
It is recommended that the microscope is equipped with a motorized XY stage, motorized epi-fluorescence turret and automatic focus system (e.g., Perfect Focus System) or equivalent supplements. When multi-well acquisition is required25, the automatic focus system will ensure a stable focus all along the experiment. Previous experience indicates that by establishing an appropriate focus offset on the Raji cells, both the movement of T cells (Video 1) and the microscope stage/chamber slide movements in XY multi-point experiments, may be compensated. This is indeed convenient for multiwell time-lapse capture.
A black and white, panchromatic and cooled charged coupled device (CDD) camera was used but higher-sensitivity, fluorescence scientific complementary metal-oxide semiconductor (sCMOS) camera is desirable, since this will decrease camera exposure times and will enhance temporal resolution. The short camera exposure times we have used (ranking form 100 ms to 500 ms) combined with the automatic fluorescence shutter allows prolonged time lapse capture (up to 24 h) with an adequate time resolution (1 frame per minute or less, for up to 16 XY positions) without significant fluorescence bleaching and/or loss in cell viability. The motorized stage allows multi-point (XY) capture and increases the chance to find and image the emerging and developing synapses in the ideal orientation, but also permits image acquisition in multiwell chamber slides when different Jurkat clones need to be simultaneously conjugated25. High numerical aperture of the objective (i.e. 60x, 1.4) is necessary in order to obtain the best results when analyzing the traffic of secretory granules.
The RAJI-SEE-Jurkat constitutes a well-established immunological synapse model that has been used by a myriad of researchers since it was originally described11. We have adapted our protocol to this model in order to properly image the early stages of IS formation. Our aim was to improve the early approaches20 previously followed to study the polarization of the MTOC and the secretory machinery towards the IS. It is remarkable that the conjugates made with this protocol produce F-actin reorganization at the synapse, configuring a canonical SMAC, concomitantly to the MVB polarized traffic25. These crucial events have been also analyzed and validated by confocal microscopy25.
Kinetic differences in polarized traffic exist among different types of IS. For example, the polarized transport of lytic granules from CTLs takes place in seconds or very few minutes, whereas several cytokine-containing vesicles from Th lymphocytes take from minutes up to several hours to finish. These temporal dissimilarities must be taken into account in advance, in order to design the best strategy and to select the most appropriate experimental and imaging approach, since for some imaging stratagems (i.e., laser scanning confocal microscopy (LSCM)), time can be a limiting factor since capture time is much higher than the appropriate time resolution (1 min or less)4. This is not a limitation when wide-field fluorescence microscopy (WFFM) is used as described in the protocol above. Since in CTLs, the polarization of MTOC towards the synapse lasts only a few minutes3,6,17, diverse specific state-of the art microscopy approaches different to that described here (but harboring higher spatial and temporal resolution) are necessary in order to properly image these synapses26,27, mainly when several microscope fields (multi-point capture) are imaged. These high-resolution, new approaches can be also utilized for imaging the synapses made by Th lymphocytes, although economical and/or logistic reasons (i.e., the core equipment required for some of these imaging techniques costs 6-7 times more than the one described here) could certainly constitute a limitation for these state of the art imaging methods4. The fact that IS made by Th lymphocytes are long-lived, and the circumstance that in Th lymphocytes the MTOC, the lymphokine-containing secretory vesicles and MVB take from several minutes up to hours to transport and dock to the IS22, makes this protocol an ideal, affordable approach for imaging the Th-APC IS.
WFFM in combination with post-acquisition image deconvolution constitutes an interesting approach and not only economic reasons support this strategy. The intrinsic poor resolution in the Z axis (the most important caveat of the technique) can be improved by using post- acquisition image deconvolution4 (compare Video 1 with Video 2). Deconvolution uses a calculation-based, image processing approach that can improve signal to noise ratio and image resolution and contrast27 up to 2 times, down to 150-100 nm in XY axis and 500 nm in the Z axis4.
The use of higher-sensitivity, high readout speed and wide dynamic range, new fluorescence sCMOS cameras will improve the quality of the images and will reduce fluorescence bleaching. The flexibility offered by the cell-to-cell conjugation protocol described here allows the combination of the described cellular approach with several state of the art microscopy techniques, both in living cells but also in fixed cells, and the expected outcome will indeed improve our knowledge of the immunological synapse.
Although we have implemented and validated the protocol by using easy-to-handle, well established cell lines, the potential the approach may allow visualization of more physiological interactions when primary T cells and different types of antigen-presenting cells (such as dendritic cells of macrophages) are used5. In this context, this protocol has been also extended and validated by using superantigen (SEB)-pulsed mouse EL-4 cell line used as an APC, to challenge primary mouse T lymphoblasts9. Indeed primary T lymphocytes, CTLs in particular, rendered more short-lived and dynamic synaptic contacts (see Supplemental Video 8 in reference9) to those seen with the SEE-Raji and Jurkat model. The variability of synaptic contact modes can best be seen for primary T-cell interactions with dendritic cells or B cells in two-dimensional in vitro tissue equivalents that can be also recorded and analyzed by using this protocol. In addition, apart from superantigens, the technique can be used to image other types of synapses. For instance, it could be used in a TCR transgenic, antigen-specific T cell model, for instance using the ovalbumin specific murine OT1/OT2 system or by transfection of T cells with antigen-specific T cell receptors. This opens a myriad of experimental possibilities for the immediate future.

The main goal of the method is to generate immunological synapse (IS) cell-to-cell conjugates formed by an antigen-presenting cell (APC) pulsed with SEE superantigens and an effector Th cell, and to register the images corresponding to the first stages of immunological synapse formation and the subsequent trafficking events (occurring both in the APC and the Th cell), that eventually will lead to polarized secretion at the IS. The establishment of the IS by T lymphocytes upon binding of their cell receptor (TCR) to antigens bound to MHC-II on the APC organizes an extremely dynamic, malleable and critical instance involved in antigen-specific, humoral and cellular immune responses1,2. The IS is defined by the formation of a special supramolecular activation complex (SMAC) pattern characterized by an actin reorganization process3. Upon IS construction by T lymphocytes with an APC, the polarization of the secretory vesicles towards the IS appears to be implicated in polarized secretion at the synaptic gap. This focused machinery appears to unambiguously supply the immune system with a superbly regulated stratagem to enhance the efficacy of critical secretory effector roles of T lymphocytes, while reducing nonspecific, cytokine-arbitrated stimulation of bystander cells, killing of irrelevant target cells and apoptotic suicide via activation-induced cell death (AICD)4.
The consequence of the IS varies on the nature of both the T lymphocyte and the APC. The synaptic contact of Th cells (typically CD4+ cells) with the APC displaying antigen-associated to MHC-II produces the activation of the T cell (cytokine secretion, proliferation, etc.) and, in some instances, apoptosis via AICD4. For cytotoxic T lymphocytes (CTLs) (principally CD8+ cells) interacting with APC presenting antigen associated to MHC-I, the outcome differs on the pre-stimulation or not of the CTLs with antigen. Thus, na&#239;ve CTLs identifying antigen-MHC-I complexes on the APC are &#34;primed&#34; to destroy target cells and divide. Primed CTLs also establish synapses with target cells (i.e., cells infected by viruses or tumor cells) producing antigen-specific cell extermination5,6.
The polarized secretion of exosomes at the immunological synapse is a developing and challenging area of research involved in relevant immune responses7. It has been demonstrated that multivesicular bodies (MVB) carrying intraluminal vesicles (ILVs) experience polarized transportation towards the IS8,9 (Video 1) upon TCR stimulation with antigen. The fusion of these MVB at the synaptic membrane induces their degranulation and the release of the ILVs as exosomes to the synaptic cleft8,10. This occurs in IS formed by Th-type Jurkat cells that were challenged with SEE superantigen-coated Raji cells acting as APC11, TCR-stimulated CD4+ lymphoblasts, and primed CTLs. Thus, synapses made by Jurkat cells constitute a valuable model to study polarized secretory traffic of exosomes. In addition, several decades of investigation have shown that many fundamental insights into TCR signaling came from studies with transformed T-cell lines, and indeed the best known of these model systems is the Jurkat leukemic T-cell line12.
The formation of a fully developed IS produces several crucial biological outcomes, including activation of Th cells, activation of na&#239;ve CTLs or target cell killing by primed CTLs, apart from anergy or AICD5. Therefore, there are two major types of secretory IS established by T lymphocytes that result in very diverse, but similarly critical, immune effector functions1,6,13. On one hand, the IS from primed cytotoxic T lymphocytes (CTLs) induces rapid polarization (ranging from seconds to few minutes) of lytic granules (called &#34;secretory lysosomes&#34;) towards the IS. The degranulation of the lytic granules induces the secretion perforin and granzymes to the synaptic cleft14, which are pro-apoptotic molecules. The secreted perforin and granzymes subsequently induce killing of the target cells15,16. CTLs develop temporary synapses, lasting only few minutes, as the target cells are murdered3,17. This is probably due to the circumstance that the optimal CTL task requires a rapid and temporary contact in order to distribute as many lethal strikes as possible to numerous target cells3,17. In contrast, Th lymphocytes such as Jurkat cells generate stable, long-standing IS (from 10-30 min up to hours), since this appears to be necessary for both directional and incessant secretion of stimulating cytokines3,17. The cytokines are also enclosed in secretory vesicles and some of them (i.e., IL-2, IFN-&#947;) experience polarized transport to the IS17 and secretion. One essential characteristic of the IS is the formation of exploratory, weak and transient contacts between the T cell and the APC (Video 1) that may produce a stronger interaction and the establishment of a mature IS, providing that the TCR identifies the cognate antigen-MHC complexes and that appropriate co-stimulatory connections are established5. Both the beginning of the initial contacts and the founding of a mature, totally productive IS, are inherently stochastic, fast and asynchronous processes5,18. Furthermore, there is a scarce frequency in the creation of cell-to-cell conjugates19, which may constitute a challenge for imaging techniques (please refer to Results and Discussion sections).
Another main challenge in examining polarization of the microtubule-organizing center (MTOC) and secretory granules in T lymphocytes is that the entire process is fast (seconds to few minutes), particularly in CTLs. Considering these facts, most early approaches confronted an end point strategy in which APC/target cells and T lymphocytes are mixed jointly and converged by low speed centrifugation, to favor cell-to-cell conjugate creation, incubated for several minutes, fixed and subsequently evaluated for the relocation of MTOC and/or secretory vesicles towards the IS20. This approach has two significant limitations: no lively trafficking data was achieved and high levels of background MTOC/secretory granules polarization were obtained, probably due to the stochastic character of IS establishment18. Furthermore, any correlation between TCR-stimulated, initial signaling events (i.e., intracellular calcium rises, actin reorganization) and secretory vesicle polarization is problematic to investigate. Thus, imperative provisions for appropriate imaging of the IS in living cells combine to enhance the cell-to-cell conjugate materialization, to synchronize the generation of the IS and, when possible, to guarantee the establishment of cellular conjugates at defined microscope XY fields and Z positions. Several strategies have been developed in order to avoid all these problems. It is out of the scope of this paper to explain these methods, their benefits and weaknesses. Please refer to the previously published reviews tackling these important points1,4,5,21.
The fact that IS made by Th lymphocytes are long-lived, and the circumstance that in Th lymphocytes the MTOC, lymphokine-containing secretory granules and MVB take from several minutes up to hours to move and dock to the IS22 makes the Th-APC IS an ideal candidate for imaging using the protocol described here.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Camera Nikon DS-QI1MC</td>
			<td>Nikon</td>
			<td>MQA11550</td>
			<td>Cooled Camera Head</td>
		</tr>
		<tr>
			<td>CMAC</td>
			<td>ThermoFisher Scientific</td>
			<td>C2110</td>
			<td>Cell tracker blue</td>
		</tr>
		<tr>
			<td>JURKAT cells</td>
			<td>ATCC</td>
			<td>ATCC TIB-152</td>
			<td>Effector T lymphocytes</td>
		</tr>
		<tr>
			<td>&#956;-Slide 8 well ibiTreat, &#956;-Slide 8 well Glass-Bottom</td>
			<td>IBIDI</td>
			<td>Cat.No: 80826, 80827</td>
			<td>Cell culture and cell imaging supports</td>
		</tr>
		<tr>
			<td>Microscope NIKON Eclipse Ti-E</td>
			<td>Nikon</td>
			<td>NIKON Eclipse Ti-E</td>
			<td>Wide-field fluorescence, fully-motorized microscope equipped with Perfect Focus System (PFS) option</td>
		</tr>
		<tr>
			<td>Microscope Stage Incubator with 3-channel manual gas mixer and gas bubbler/ humidity module</td>
			<td>OKOLAB</td>
			<td>H201-NIKON-TI-S-ER</td>
			<td>Cell culture atmosphere</td>
		</tr>
		<tr>
			<td>Raji Cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-86</td>
			<td>APC</td>
		</tr>
		<tr>
			<td>RPMI medium GIBCO</td>
			<td>ThermoFisher Scientific</td>
			<td>21875034</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>Streptococcus Enterotoxin E (SEE)</td>
			<td>Toxin Technology, Inc</td>
			<td>EP404</td>
			<td>Bacterial Toxin</td>
		</tr>
		<tr>
			<td>Software Huygens Essential</td>
			<td>SVI</td>
			<td>Huygens Essential</td>
			<td>Image Deconvolution software</td>
		</tr>
		<tr>
			<td>Software Image J</td>
			<td>NIH</td>
			<td>Image J</td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>Software Nikon NIS-AR</td>
			<td>Nikon</td>
			<td>NIS-Elements AR</td>
			<td>Image capture and analysis software</td>
		</tr>
	</tbody>
</table>

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.

We have followed the described protocol in order to generate Jurkat-Raji immune synapse conjugates and to properly image the early stages of IS formation. Our aim was to improve the early approaches20 previously followed to study the polarization of the MTOC and the secretory machinery towards the IS. These approaches were based on an end point strategy that did not allow imaging of the IS formation or the early synaptic events, since in these strategies the IS formation obligatory occurred in the pellet of mixed, centrifuged cells, but not on a microscope. Our protocol was designed to avoid this main caveat, since the approach was based on the use of an appropriate cell concentration (Steps 2 and 3), to favor the formation of the cell-to-cell IS conjugates on the own microscope chamber slides (8 micro well chamber slide),mounted on the pre-heated microscope stage incubator (in Step 4). This strategy induces the IS formation, simultaneously to the time-lapse imaging capture (Video 1).
Figure 1 represents synaptic Jurkat-Raji conjugates that were obtained following the protocol (step 5.2). The image represents the first frame from a representative, time-lapse experiment. 2 x 105 Raji cells and 2 x 105 Jurkat cells were added to a 1 cm2 well. The upper panel shows transmittance channel, the middle panel consists of CMAC (blue) channel (Raji cells), and the lower panel shows both transmittance plus CMAC merged channels. Yellow arrows label some synaptic conjugates, as a reference, whereas green arrows indicate synaptic conjugates made by one Jurkat cell and several Raji cells (complex conjugates). Decreasing cell concentrations (105 or less cells in the 1 cm2 well) will circumvent the formation of complex cellular conjugates, but there may not be enough cell conjugates for subsequent analysis of polarized traffic (see below), that in turn will decrease also the chances to find and to image emerging synaptic conjugates.
Figure 2 represents screenshots corresponding to the imaging parameters used for the simultaneous capture of the two different fluorochromes (CMAC and GFP-CD63) by using appropriate software (i.e., NIKON NIS_AR) in a representative time lapse experiment corresponding to Video 1.
Video 1 (Immunological synapses, raw) represents a Jurkat T lymphocyte expressing GFP-CD63 (a marker of multivesicular bodies-MVB, green vesicles) and the formation of a double synapse between 1 Jurkat cell and 2 Raji cells labelled with CMAC, in blue (one of them undergoes engulfment). The simultaneous movement of MVB inside the Jurkat cell towards synapse areas was recorded (capture frame rate = 1 frame every 20 seconds for GFP-CD63; video reproduction speed = 2 frames per s). The synaptic conjugates were obtained and imaged following the protocol described above (Step 6.2), which allowed imaging of emerging synapses (Video 1). The simultaneous capture of both GFP-CD63 and CMAC fluorescence channels was performed by using an appropriate software (i.e., NIS-AR, Table of Materials). The video represents the raw data from a representative, time-lapse experiment. The automatic focus system was defined with an appropriate offset with respect to the Raji cells bound to the glass bottom, to assure a stable focus along the experiment.
Video 2 (immunological synapses, after deconvolution) corresponds to Video 1, but the deconvolution of the GFP-CD63 fluorescence channel images was performed by employing an appropriate deconvolution software (i.e., Huygens), using the &#34;wide field&#34; optical option and the proper optical parameters (step 8). This deconvoluted channel was subsequently merged to the CMAC, raw channel. The improvement of both signal-to-noise ratio and sharpness of the image is evident. The deconvolution was performed post-acquisition, as described above. For specific details regarding the deconvolution software please refer to4.
Video 3 (immunological synapse, after fixing and immunofluorescence staining) represents a Z-stack (z-step size = 0.8 &#181;m, 5 frames) of a fixed IS conjugate after the step 6 of the protocol (acetone fixation). After fixation, immunofluorescence was performed following standard protocols25 by using Phalloidin to visualize the F-actin (green), anti-CD63 to visualize MVB (magenta), anti-&#947;-tubulin to visualize MTOC (red). CMAC (blue) labels the Raji cell. Subsequently the conjugate was imaged by epifluorescence and several channels deconvoluted using the &#34;wide field&#34; optical option and the proper optical parameters (step 7). The deconvoluted channels were subsequently merged to the CMAC, raw channel, as indicated in the different panels (CMAC plus Transmittance-TRANS, CMAC plus Phallodin, CMAC plus anti-CD63 and CMAC plus anti-&#947;-tubulin, respectively). White arrow labels the synapse, whereas green arrow labels the MVB and the yellow arrow labels the MTOC. The detailed quantification of MVB and MTOC polarization has been explained elsewhere25.
The approach presented here involves the formation of cell-to-cell conjugates and, simultaneously, the time-lapse acquisition by wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This tactic improved the signal-to-noise ratio (SNR) of the images, enhanced their temporal resolution, and allowed the synchronized acquisition of several fluorochromes in emerging synaptic conjugates4. In addition, the protocol is well matched with subsequent end point cell fixation methods (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses25 (Video 3). This protocol is also compatible with laser scanning confocal microscopy and other state-of-the-art microscopy techniques.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60312/60312fig1.jpg" /> 
Figure 1: Representative double-size microscopy field of synaptic conjugates. The image represents the first frame from a representative, time-lapse experiment following the protocol. The upper panel shows the transmittance channel, the middle panel CMAC channel (Raji cells) and the lower panel both merged channels. Yellow arrows label some synaptic conjugates, as a reference. Green arrows indicate complex synaptic conjugates (i.e., one Jurkat cell establishing synapses with more than one Raji cell). Captured with a 40x EWD (0.6 NA) objective. <a href="https://www.jove.com/files/ftp_upload/60312/60312fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60312/60312fig2a.jpg" /> 
Figure 2: Time lapse microscope settings. The image corresponds to several screenshots corresponding to the imaging parameters used for the simultaneous capture of the two different fluorochromes (CMAC and GFP-CD63) using appropriate software (i.e., NIKON NIS_AR) in a representative time lapse experiment corresponding to Video 1. Each frame for GFP-CD63 channel was captured every 20 s. Only one frame for a UV channel out of eight frames for GFP channel was captured in order to maintain cell viability along the experiment. <a href="https://www.jove.com/files/ftp_upload/60312/60312fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/60312/60312vid1.jpg" /> 
Video 1: Immunological synapses made by a Jurkat cell expressing GFP-CD63, raw data. Raji B cells labelled with cell tracker blue (CMAC, blue) were pulsed with SEE for 30 min and synapses with Jurkat cells expressing GFP-CD638 were formed. Time-lapses corresponding to GFP-CD63 and CMAC channels were captured (20 s per frame; video reproduction speed = 2 frames per s) and a representative example is shown. Captured with a 60x PLAN APO (1.4 NA) objective. <a href="https://www.jove.com/files/ftp_upload/60312/Video_1.mov" target="_blank">Please click here to view this video (Right click to download).</a>
<img alt="Video 2" src="/files/ftp_upload/60312/60312vid2.jpg" /> 
Video 2: Immunological synapses made by a Jurkat cell expressing GFP-CD63, after deconvolution. Same as Video 1, but images were deconvoluted using a deconvolution software. The improved signal-to noise ratio and the enhanced sharpness, due to the elimination of contaminant out of focus fluorescence, is evident. <a href="https://www.jove.com/files/ftp_upload/60312/Video_2.mov" target="_blank">Please click here to view this video (Right click to download).</a>
<img alt="Video 3" src="/files/ftp_upload/60312/60312vid3v2.jpg" /> 
Video 3: Fixed and immunostained immunological synapse. The image shows a representative fixed synaptic conjugate after Step 7 of the Protocol and subsequent immunofluorescence. CMAC (blue) labels raji cells, transmittance (TRANS) to show the synaptic conjugate (white arrow), Phalloidin labels F-actin (green), anti-CD63 labels MVB (magenta, green arrow) and anti-&#947;-tubulin labels MTOC (red, yellow arrow), respectively. These channels were imaged by epifluorescence, deconvoluted and merged as indicated. The video includes a Z-stack (z-step size = 0.8 &#956;m, 5 frames). White arrow labels the synaptic contact area, whereas the green arrow labels the MVB and the yellow arrow labels the MTOC. <a href="https://www.jove.com/files/ftp_upload/60312/Video_3.mov" target="_blank">Please click here to view this video (Right click to download).</a>

Watch this Scientific Journal Video about Imaging the Human Immunological Synapse at JoVE.com

Imaging the Human Immunological Synapse

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) ...

Research

JoVE Journal

Immunology and Infection

13.9K Views.  CSIC-Universidad Autónoma de Madrid. 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).

Video: Imaging the Human Immunological Synapse

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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

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

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

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

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

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

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

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

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

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

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

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

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

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

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

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

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

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

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

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

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