The work was funded by the German research council (DFG) with grants No. SFB-938-M and SA 393/3-4.

1. Preparation of Pan-leukocytes
<ol>
	<li>Draw 1 mL of peripheral blood from a healthy donor (or patient) in a heparinized syringe. Make sure to have approval by the responsible ethics committee for the blood donation.</li>
	<li>Mix 1 mL of human peripheral blood with 30 mL of ACK lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA, pH 7.0) in a 50 mL tube and incubate for 8 min at room temperature.</li>
	<li>Fill the tubes with PBS and centrifuge at 300 x g for 6 min. Aspirate the supernatant and resuspend the pellet in 30 mL of ACK lysis buffer.</li>
	<li>Repeat steps 1.2 and 1.3 until the supernatant is clear. Finally, wash the cells in PBS, centrifuge at 300 x g for 6 min at room temperature, and resuspend the cell pellet in 2 mL of culture medium (RPMI1640 + 10% FCS). Incubate the cells at 37 &#176;C for 60 min.</li>
</ol>
2. Loading of Raji Cells with SEB
<ol>
	<li>Prepare two 15 mL Falcon tubes with 1.5 x 106 Raji cells per tube. Spin down the cells (300 x g for 6 min at room temperature) and discard the supernatant.</li>
	<li>Resuspend the cells in residual medium (about 50&#8211;100 &#181;L), add 1.9 &#181;L (1.9 &#181;G) SEB, and incubate at room temperature for 15 min. Add 5 mL of culture medium, spin down the cells (300 x g for 6 min at room temperature), and resuspend the pellet in culture medium (RPMI1640 + 10% FCS) at a density of 1 x 106 cells/mL.</li>
</ol>
3. Induction of Immune Synapses and Staining Protocol
<ol>
	<li>Pipette 500 &#181;L of the preparation of SEB-loaded Raji cells into a FACS tube and 500 &#181;L of the preparation of unloaded Raji cells into another FACS tube. Add 650 &#181;L of pan-leukocytes to each tube and spin down the cells (300 x g for 10 min at room temperature). Discard the supernatant and resuspend the pellet in 150 &#181;L of culture medium (RPMI1640 + 10% FCS). Incubate at 37 &#176;C (typically for 45 min).</li>
	<li>Gently vortex the cells (10 s at 1,000 rpm) and add 1.5 mL of paraformaldehyde (1.5%) during the vortex to fix the cells. Stop the fixation by adding 1 mL of PBS + 1% BSA. Pellet the cells (300 x g for 10 min at room temperature and resuspension the cell pellet in 1 mL of PBS + 1% BSA after incubating at room temperature for 10 min.</li>
	<li>Pellet (300 x g for 10 min at room temperature) and resuspend the cells in 100 &#181;L of PBS + 1% BSA + 0.1% saponin for 15 min at room temperature to permeabilize the cells (96-well plate, U-shaped).</li>
	<li>Wash the cells in PBS + 1% BSA + 0.1% saponin with centrifugation (300 x g for 10min at room temperature) and resuspend the cell pellet in 50 &#181;L of PBS + 1% BSA + 0.1% saponin containing fluorophore-labelled antibodies or compounds (CD3-PE-TxRed (1:30), Phalloidin-AF647 (1:150), and DAPI (1:3,000)).</li>
	<li>Incubate the cells at room temperature in the dark for 30 min. Wash the cells 3 times by adding 1 mL of PBS + 1% BSA + 0.1% saponin. Centrifuge at 300 x g for 10 min at room temperature. Re-resuspend the cells in 60 &#181;L of PBS for imaging flow cytometry.</li>
</ol>
4. Image Acquisition Using a Flow Cytometer
NOTE: The following image acquisition procedure and data analysis are based on imaging flow cytometry using software such as imagestream (IS100), INSPIRE, and IDEAS. However, other flow cytometers and analysis software can also be used.
<ol>
	<li>Open the analysis software on the computer connected to the imaging flow cytometer and click on Initialize Fluidics of the Instrument menu. Apply the beads on the right port when prompted to do so.</li>
	<li>Load the default template from the File menu and click on Run/Setup. Choose Beads from the View dropdown menu.</li>
	<li>Adjust the bright-field illuminator by clicking on Set Intensity if the indicated value is below 200.</li>
	<li>Run the calibration and test routine in the Assist tab by clicking on Start All.</li>
	<li>Click on Flush/Lock/Load and apply the samples in the left port when prompted to do so. After loading the cells in the flow cytometer, open the Cell Classifier and adjust the values as follows: peak intensity upper limit at 1,022 for each channel, peak intensity lower limit at 50 for channel 2 (DAPI) and channel 5 (CD3-Pe-TxR), area lower limit at 50 for channel 1 (side scatter), and upper limit at 1,500.</li>
	<li>Change the excitation laser power to 405 nm (15 mW), 488 nm (200 mW), and 647 nm (90 mW) in the Setup tab.</li>
	<li>Switch the View dropdown menu between Cells and Beads to evaluate the cell classifier and laser power adjustments. 
	NOTE: Make sure that all cells and cell couples are found the Cell View and that cell clumps, debris, and images with saturated pixels are found in the Debris View by changing the cell classifiers and/or the excitation laser powers.</li>
	<li>Define the sample name and the amount of images to acquire (15,000&#8211;25,000 for samples and 500 for compensation controls) in the Setup tab. Click on Run/Setup to start the acquisition.</li>
</ol>
5. Data analysis
<ol>
	<li>Transfer the raw image files (.rif) to the data analysis computer and open the analysis software.</li>
	<li>Produce a compensation matrix following the instructions of the Compensation dropdown. Save the compensation matrix as comp_Date.ctm.</li>
	<li>Open a sample raw image file (.rif) and apply the comp_Date.ctm in the window that appears to produce the compensated image files (.cif) and the default data analysis file (.daf).</li>
	<li>Open the compensated image file. Convert the images to color mode and adjust the lookup tables to obtain optimal visible colors in the Image Gallery Properties toolbar. Obtain an RGB-merged image using the Composite tab of the Image Gallery Properties toolbar.</li>
	<li>Open the Mask Manager from the Analysis dropdown. Create masks to define the T cells and the immune synapse, as follows:
	<ol>
		<li>Select the T-cell mask: &#34;(Fill(Threshold_Ch05, 60).&#34; Select the valley mask: &#34;Valley(Ch02,3).&#34; Select the T-cell synapse mask: &#34;T-cell mask AND Valley(M02,Ch02, 3).&#34;</li>
	</ol>
	</li>
	<li>Open the Feature Manager from the Analysis dropdown. Calculate the following features:
	<ol>
		<li>For the total CD3 expression in T cells, select &#34;Intensity_T-cells_Ch5.&#34; For the total amount of F-actin in the T cells, select &#34;Intensity_T-cells_Ch6.&#34; For CD3 expression in the immune synapse, select &#34;Intensity_T-cell synapse_Ch5.&#34; For the amount of F-actin in the immune synapse, select &#34;Intensity_T-cell synapse_Ch6.&#34;</li>
		<li>To calculate the T-cell area, select &#34;Area_T-cells.&#34; To calculate the T-cell immune synapse area, select &#34;Area_T-cell synapse.&#34;</li>
	</ol>
	</li>
	<li>Determine the F-actin and CD3 enrichment in the immune synapse by using the equation in the Feature Manager: 
	<img alt="Equation" src="/files/ftp_upload/55345/55345eq1.jpg" /></li>
	<li>Apply the following gating strategy by using histograms and dot plots from the Analysis area (for further details, see References 17 and 19):
	<ol>
		<li>Discard out-of-focus cells by plotting the &#34;Gradient RMS_M2_Ch2&#34; in a histogram; set the threshold at 15.</li>
		<li>Plot the SSC versus CD3 intensity in a dot plot. Set a gate on CD3-positive events.</li>
		<li>Plot the &#34;Aspect ratio&#34; of M02 (DAPI stain) versus the area of M02 (Dapi stain). Gate on T-cell singlets and cell couples accordingly. Correct for true cell couples using the area of the synapse mask, as described previously17,19.</li>
		<li>Determine the amount of F-actin in T-cell singlets and T cells of T-cell/APC couples and the percent of F-actin in the immune synapse.</li>
	</ol>
	</li>
</ol>

<ol><li>Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD28+Costimulation%3A+From+Mechanism+to+Therapy%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">CD28 Costimulation: From Mechanism to Therapy.</a> Immunity. 44 (5), 973-988 (2016).</li>
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<li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+the+immunological+synapse+by+dual+color+time-gated+stimulated+emission+depletion+%28STED%29+nanoscopy%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.</a> J Vis Exp. (85), (2014).</li>
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<li>Markey, K. A., Gartlan, K. H., Kuns, R. D., MacDonald, K. P., Hill, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+the+immunological+synapse+between+dendritic+cells+and+T+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Imaging the immunological synapse between dendritic cells and T cells.</a> J Immunol Methods. 423, 40-44 (2015).</li>
<li>Bouma, G., Burns, S. O., Thrasher, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wiskott-Aldrich+Syndrome%3A+Immunodeficiency+resulting+from+defective+cell+migration+and+impaired+immunostimulatory+activation%5BTitle%5D)+AND+%22Immunobiology%22%5BJournal%5D)">Wiskott-Aldrich Syndrome: Immunodeficiency resulting from defective cell migration and impaired immunostimulatory activation.</a> Immunobiology. 214 (9-10), 778-790 (2009).</li>
<li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunological+synapse%3A+a+molecular+machine+controlling+T+cell+activation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285 (5425), 221-227 (1999).</li>
<li>Dustin, M. L., Depoil, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+insights+into+the+T+cell+synapse+from+single+molecule+techniques%5BTitle%5D)+AND+%22Nature+reviews.+Immunology%22%5BJournal%5D)">New insights into the T cell synapse from single molecule techniques.</a> Nature reviews. Immunology. 11 (10), 672-684 (2011).</li>
<li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+views+of+the+human+NK+cell+immunological+synapse%3A+recent+advances+enabled+by+super-+and+high-resolution+imaging+techniques%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">New views of the human NK cell immunological synapse: recent advances enabled by super- and high-resolution imaging techniques.</a> Front Immunol. 3, 421(2012).</li>
<li>Yokosuka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat Immunol. 6 (12), 1253-1262 (2005).</li>
<li>Wabnitz, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sustained+LFA-1+cluster+formation+in+the+immune+synapse+requires+the+combined+activities+of+L-plastin+and+calmodulin%5BTitle%5D)+AND+%22European+journal+of+immunology%22%5BJournal%5D)">Sustained LFA-1 cluster formation in the immune synapse requires the combined activities of L-plastin and calmodulin.</a> European journal of immunology. 40 (9), 2437-2449 (2010).</li>
<li>Wabnitz, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((L-plastin+phosphorylation%3A+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li>
<li>Wabnitz, G. H., Nessmann, A., Kirchgessner, H., Samstag, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((InFlow+microscopy+of+human+leukocytes%3A+A+tool+for+quantitative+analysis+of+actin+rearrangements+in+the+immune+synapse%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">InFlow microscopy of human leukocytes: A tool for quantitative analysis of actin rearrangements in the immune synapse.</a> J Immunol Methods. , (2015).</li>
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<li>Wabnitz, G. H., Samstag, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiparametric+Characterization+of+Human+T-Cell+Immune+Synapses+by+InFlow+Microscopy%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Multiparametric Characterization of Human T-Cell Immune Synapses by InFlow Microscopy.</a> Methods Mol Biol. 1389, 155-166 (2016).</li>
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</ol>

The authors have nothing to disclose.

The workflow presented here enables the quantification of immune synapses between human T cells (ex vivo) and APCs. Notably, erythrocyte-lysed pan-leukocytes were used as T-cell sources, making T-cell purification steps dispensable. The B-cell lymphoma cell line Raji served as surrogate APCs. This bears significant advantages, since it allows comparisons between blood donors of the T-cell side of the immune synapse. Furthermore, autologous DCs are hardly available directly from peripheral human blood. The production of monocyte-derived dendritic cells (moDCs) takes several days, making the application to clinical studies challenging. However, imaging flow cytometry and the analysis strategy presented here can be applied to other APCs (e.g., transdifferentiated neutrophils)20. Moreover, it was demonstrated that the immune synapse between CD4 T cells and ex vivo DCs9 or B cells21 can be assessed for mice using imaging flow cytometry. Thus, this method can be broadened to assess APC function in addition to the T-cell side of the immune synapse.
The most critical steps of this methodology are mixing the pan-leukocytes and APCs in a small volume and performing the correct gating strategy to exclude false-positive events while at the same time minimizing the loss of true immune synapses. While we describe the measurement of CD3 and F-actin accumulation, this method is not restricted to these proteins and can be expanded to other surface receptors, such as LFA-117,19. Moreover, the addition of fluorophore-labelled antibodies to identify T-cell subgroups (e.g., CD4, CD8, CD45RA, or chemokine receptors) would allow for the exploration of the nature of T cells displaying a certain immune synapse phenotype. Importantly, the amount of blood that is necessary for such an analysis is low (maximum: 1 mL). While the first generation of imaging flow cytometers, as used here (IS100), have an image acquisition rate of about 100 cells/s, the third generation of imaging flow cytometers (IsXMKII) allows much higher image acquisition rates (up to 2,000 cells/s using a 40x objective). Thus, the actual workflow, from drawing the blood to data analysis, requires a considerably short time period (i.e., one day). Notably, while the presented image analysis is based on imaging flow cytometry, cell preparation and the induction of immune synapses (i.e., the first part of the workflow) can be applied to other imaging techniques22.
In contrast to flow cytometry, imaging flow cytometry-obtained dot plots contain information about protein localization in addition to protein expression data. One strength of imaging flow cytometry is that cells (or cell couples) of any selected gate can be evaluated by eye; the gate boundaries can be adjusted accordingly, simply by clicking on the dots and inspecting the corresponding image. In our experiments, we found that 30% protein enrichment is a reliable value to consider the respective cell couple as having the protein of interest enriched. Once the gates are finally set, the features and gating strategy are saved in a separate file (.ast) and can be applied to each following sample to assure an unbiased evaluation.
The weaknesses of imaging flow cytometry are the limitation in resolution (0.5 &#181;m using a 40x objective) and the fact that only one focus plane can be analyzed. Therefore, this method is not suitable to analyze the fine structure of the immune synapse and the occurrence of microclusters14. To analyze such architecture-related aspects of the immune synapse, alternate techniques, such as confocal, TIRF, or super-resolution microscopy, are needed. The strength of imaging flow cytometry is the amount of images that can be acquired per sample (up to 25,000). Notably, each image is segmented, which means that the background is knocked out, and there is usually only one solitary cell or cell couple per image. These characteristics of imaging flow cytometry form the basis for automated analysis at the single-cell level. Moreover, the high amount of cells that are analyzed per sample allow for the reliable identification of rare events (i.e., subpopulations or cell couples with a frequency below 1%). Importantly, the workflow presented here can be applied to the study of immune synapse formation in ex vivo T-cells in pan-leukocytes from patients suffering from immune-related diseases (e.g., primary immunodeficiency disorders).

T cells are major regulators of the adaptive immune system and are activated through antigenic peptides that are presented in the context of major histocompatibility complexes (MHC). Full T-cell activation requires two signals, the competence signal via the antigen-specific T-cell receptor (TCR)/CD3 complex and the costimulatory signal via accessory receptors. Both signals are generated through the direct interaction of T cells with antigen-presenting cells (APCs). Mature APCs provide the competence signal for T-cell activation through MHC-peptide complexes, and they express costimulatory ligands (e.g., CD80 or CD86) to assure the progression of T-cell activation1. One important function of costimulation is the rearrangement of the actin cytoskeleton2,3,4. The cortical F-actin is relatively static in resting T cells. T-cell stimulation through antigen-bearing APCs leads to a profound rearrangement of the actin cytoskeleton. Actin dynamics (i.e., fast actin polymerization/depolymerization circles) enable the T cells to create forces that are used to transport proteins or organelles, for example. Moreover, the actin cytoskeleton is important for developing a special contact zone between T cells and APCs, called the immune synapse. Due to the importance of the actin cytoskeleton to the immune synapse, it has become essential to develop methods to quantify changes in the actin cytoskeleton of T cells5,6,7,8,9.
By means of actin cytoskeletal aid, surface receptors and signaling proteins are segregated in supramolecular activation clusters (SMACs) within the immune synapse. The stability of the immune synapse is assured by the binding of receptors to F-actin bundles that increase the elasticity of the actin cytoskeleton. Immune synapse formation has been shown to be critical for the generation of the adaptive immune responses. The detrimental effects of a defective immune synapse formation in vivo were first realized in patients suffering from Wiskott Aldrich Syndrome (WAS), a disease in which actin polymerization and, concomitantly, immune synapse formation are disturbed10. WAS patients can suffer from eczema, severe recurrent infections, autoimmune diseases, and melanomas. Despite this finding, it is currently not known whether immune synapse formation differs in the T cells of healthy individuals and patients suffering from immune defects or autoimmune diseases.
Fluorescence microscopy, including confocal, TIRF, and super-resolution microscopy, were used to uncover the architecture of the immune synapse11,12,13,14. The high resolution of these systems and the possibility of performing live-cell imaging enables the collection of exact, spatio-temporal information about the actin cytoskeleton and surface or intracellular proteins in the immune synapse. Many results, however, are based on the analysis of only a few tens of T cells. Moreover, T cells must be purified for these types of fluorescence microscopy. However, for many research questions, the use of unpurified cells rather than the highest-possible resolution is of the utmost importance. This is relevant if T cells from patients are analyzed, since the amount of donated blood is limited and there might be the need to process many samples in parallel.
We established microscopic methods that allow the analysis of the actin cytoskeleton in the immune synapse in the human system15,16,17. These methods are based on imaging flow cytometry, also called In-Flow microscopy18. As a hybrid between multispectral flow cytometry and fluorescence microscopy, imaging flow cytometry has its strengths in analyzing morphological parameters and protein localization in heterogeneous cell populations, such as pan-leukocytes from the peripheral blood. We introduced a methodology that enables us to quantify F-actin in T-cell/APC conjugates of human T cells from whole-blood samples, without the need of time-consuming and costly purification steps17. The technique presented here comprises the whole workflow, from getting the blood sample to the quantification of F-actin in the immune synapse.

<table><tbody><tr><td>&gt;Multifuge 3 SR</td><td>Heraeus</td><td></td><td></td></tr><tr><td>RPMI 1640</td><td>LifeTechnologies</td><td>#11875085</td><td>500 mL</td></tr><tr><td>FCS</td><td>Pan Biotech#3302-P101102</td><td></td><td></td></tr><tr><td>Polystyrene Round Bottom Tube</td><td>Falcon</td><td>#352054</td><td>5 mL</td></tr><tr><td>Kulturflasche</td><td>Thermo Scientific</td><td>#178883</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8662</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Roth</td><td>#8076.3</td><td></td></tr><tr><td>Saponin</td><td>Sigma</td><td>S7900</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>#16005</td><td></td></tr><tr><td>FACS Wash Saponin</td><td></td><td>PBS 1% BSA 0.15 Saponin</td><td></td></tr><tr><td>ReaktiosgefaB</td><td>Sarstedt</td><td>72.699.002</td><td>0.5 mL</td></tr><tr><td>Speed Bead</td><td>Amnis</td><td>#400041</td><td></td></tr><tr><td>Minishaker MS1</td><td>IKA Works</td><td>&amp;nbsp;MS1</td><td></td></tr><tr><td>Mikrotiterplatte</td><td>Greiner Bio One</td><td>#650101</td><td>96U</td></tr><tr><td>Enterotoxin SEB</td><td>Sigma Aldrich</td><td>S4881</td><td></td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td>1:3000</td></tr><tr><td>CD3-PeTxR</td><td>Invitrogen</td><td>MHCD0317</td><td>1:30</td></tr><tr><td>Phalloidin-AF647</td><td>Molecular Probes</td><td>A22287</td><td>1:150</td></tr><tr><td>IS100</td><td>Amnis</td><td></td><td>Imaging flow cytometer</td></tr><tr><td>IDEAS</td><td>Amnis</td><td></td><td>Software</td></tr><tr><td>INSPIRE</td><td>Amnis</td><td></td><td>Software</td></tr></tbody></table>

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.

A major goal of the method described here is the quantification of protein enrichment (e.g., F-actin) in the immune synapse between surrogate APCs (Raji cells) and T cells in unpurified pan-leukocytes taken from low-volume (1 mL) human blood samples. The screenshot in Figure 1 gives an overview of the critical gating strategy of this method. It shows the image gallery on the left and the analysis area on the right (Figure 1). The image gallery shows the &#34;In Focus&#34; gate. The depicted images are mainly granulocytes and two T-cell/APC couples. F-actin and CD3 are enriched in cell couple number 30272. The gating strategy to quantify the amount of cell couples with such enrichment is shown in the analysis area and is described in the protocol (see step 5.8) or elsewhere17,19. The last dot plot in the analysis area displays the amount (percent protein) of CD3 (x-axis) and F-actin (y-axis) in the immune synapse. If more than 30% of the protein was located within the immune synapse mask, it was considered as protein enrichment in the immune synapse20. Dot plots containing enrichment data were used to produce a typical final result (Figure 2A). The imagery on the left shows sample images of T-cell/APC conjugates, with low amounts of CD3 and F-actin in the immune synapse, whereas on the right, T-cell/APC conjugates are depicted with a strong enrichment of CD3 and F-actin. The amount of CD3 at the immune synapse (percent protein) is plotted on the x-axis, and the amount of F-actin at the immune synapse is plotted on the y-axis. The percentage in the gate represents the amount of T cells that have an enrichment of CD3 and F-actin at the immune synapse in the presence of a superantigen. In the absence of superantigen, 15% percent of the T cells showed an enrichment at the immune synapse of both CD3 and F-actin. The amount of cells increased to 29% in the presence of superantigen. A single quantification of CD3 enrichment (Figure 2B) or F-actin enrichment (Figure 2C) is shown in the presence and absence of superantigen. These results show that, in the absence of superantigen, 18.3 &#177; 3.5% and, in the presence of superantigen, 34.3 &#177; 4.0% of the total F-actin amount in the cells were accumulated at the immune synapse. Interestingly, there was already CD3 accumulation in the absence of superantigen (16.6 &#177; 2.1% of the total CD3 amount), which was significantly increased by the addition of superantigen (24.6 &#177; 3.0% of the total CD3 amount). This method allows the quantification of how much protein is accumulated at the immune synapse between T cells and APCs.
<img alt="Figure 1" src="/files/ftp_upload/55345/55345fig1.jpg" /> 
Figure 1: Gating strategy for the identification of immune synapses in pan-leukocyte preparations. The figure shows a screenshot of the software and contains a complete analysis workflow for the evaluation of CD3 and F-actin enrichment in the immune synapse of T cells conjugated to APCs in the presence of SEB. Images are displayed in the image gallery on the left (Ch2 = DAPI; Ch5 = CD3-PETxR; CH6 = Phalloidin AF647; and Merge = Combined image containing Ch2, Ch5, and Ch6). The software provides an image display toolbar to adjust for lookup tables, mask display, and color/grayscale mode. The right portion of the screenshot shows the analysis area and analysis toolbar. The analysis area contains histograms and dot plots as follows: 1) Histogram to find cells in focus according to the gradient RMS feature of the DAPI staining. 2) Gating on T cells according to the expression of CD3 (Intensity_MC_Ch05, x-axis) and the side scatter profile (Intensity_MC_Ch01, y-axis). 3) Gating on potential T-cell/APC couples according to the aspect ratio of the DAPI stain (Aspect ratio_M02, y-axis) and the side scatter profile (Intensity_MC_Ch01, x-axis). 4) Gating on true T-cell/APC couples according to the area synapse mask (x-axis) and the area of the CD3 stain (y-axis). 5) Gating on mature immune synapses defined by enrichment (&#62;30% protein content in the interface) of CD3 (x-axis) and F-actin (y-axis) in the immune synapse. <a href="http://cloudflare.jove.com/files/ftp_upload/55345/55345fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55345/55345fig2.jpg" /> 
Figure 2: Imaging flow cytometry data on CD3 and F-actin enrichment in the immune synapse.&#160;(A) The dot plots in the center show the percentage of CD3 (x-axis) and F-actin (y-axis) in the immune synapse. T-cell/APC conjugates with a mature immune synapse in the absence (upper part) or presence (lower part) of SEB are depicted. The images on the left show T-cell/APC couples with a low degree of CD3 and F-actin enrichment, whereas the images on the right display T-cell/APC couple with a high degree of CD3 and F-actin enrichment (mature immune synapse). Scale bar = 10 &#181;m (lower left). The result is representative of three independent experiments. (B-C) Mean CD3 (B) or F-actin (C) enrichment in the immune synapse in the presence or absence of SEB is shown as percent protein (n = 3; SE). <a href="http://cloudflare.jove.com/files/ftp_upload/55345/55345fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry at JoVE.com

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

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

11.5K Views.  University of Heidelberg. 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.

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

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

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

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

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Neuroscience

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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 ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description 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) ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Analysis of Immune Cells in Mouse Nervous Tissues Using Flow Cytometry

Source:&#160;Hidmark, A. S.,&#160;et al.&#160;Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow ...

JoVE Encyclopedia of Experiments

Neuroimaging

Synaptic & Cellular Imaging

Analysis of Immune Synapses between Human T Cells and SEB-Loaded Raji Cells using Imaging Flow Cytometry

Source:&#160; Wabnitz, G. et al., <a href="https://app.jove.com/v/55345">Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry.</a> J. Vis. Exp. (2019)
This video analyzes the immune synapse formation between T cells in unpurified pan-leukocyte preparations and Staphylococcus aureus enterotoxin B, SEB-loaded Raji cells. CD3 and F-actin enrichment is observed in the immune synapse using imaging flow cytometry.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Pan-leukocytes

	...