analysis of immune synapses between human t cells and seb-loaded raji cells using imaging flow cytometry

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

Begin by mixing one milliliter of freshly drawn human peripheral blood with 30 milliliters of ACK lysis buffer in a 50-milliliter conical tube. After eight minutes at room temperature, fill the tube with PBS for a centrifugation wash, and resuspend the pellet in two milliliters of culture medium for a 60-minute incubation at 37 degrees Celsius.
At the end of incubation, add 5 x 105 Staphylococcus aureus enterotoxin B or SEB loaded or unloaded Raji cells in 500 microliters of culture medium into individual FACS tubes, followed by 650 microliters of pan-leukocytes. Spin down the co-cultures, and resuspend the pellets in 150 microliters of fresh culture medium for a 45-minute incubation at 37 degrees Celsius.
Then, gently vortex the cells for 10 seconds at 100 RPM while adding 1.5 milliliters of 1.5% paraformaldehyde. After 10 minutes at room temperature, stop the fixation with one milliliter of PBS supplemented with 1% BSA, and collect the cells by centrifugation. Resuspend the pellets in 100 microliters of PBS plus BSA and 0.1% saponin, and add the cell samples to two individual wells of a 96-well U-bottom plate.
After 15 minutes at room temperature, centrifuge the plate and resuspend the pellets in 50 microliters of PBS plus BSA and saponin, containing the fluorophore-conjugated antibodies or compounds of interest for a 30-minute incubation in the dark at room temperature. At the end of the incubation, wash the cells three times in 150 microliters of PBS plus BSA and saponin, and resuspend the pellets in 60 microliters of PBS.
To image the cells by flow cytometry, open the analysis software, check the liquid level, and click Start Up in the instrument menu. Next, click Load, and load the samples into the port when prompted.
Under the Illumination tab, change the excitation laser power to the appropriate nanometer lengths. Then, adjust the gates for channels 4 and 11, and adjust the area for channel 1.
To evaluate the gating and laser power adjustments, switch the View menu to the respective gate, and adjust the gates and laser powers until the cells and cell couples are visible. Then, in the File Acquisition tab, define the sample name, and the number of images to acquire, and the appropriate gate for CD3 staining, and click Acquire to begin the acquisition.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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 10 min at room temperature) and resuspend the cell pellet in 50 &#181;L of PBS + 1% BSA + 0.1% saponin containing fluorophore-labeled 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 in 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 in 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 number 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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>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>Dulbecco&#39;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>Speed Bead</td>
			<td>Amnis</td>
			<td>#400041</td>
			<td></td>
		</tr>
		<tr>
			<td>Minishaker MS1</td>
			<td>IKA Works</td>
			<td>MS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</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>One to three thousand ratio</td>
		</tr>
		<tr>
			<td>CD3-PeTxR</td>
			<td>Invitrogen</td>
			<td>MHCD0317</td>
			<td>One to thirty ratio</td>
		</tr>
		<tr>
			<td>Phalloidin-AF647</td>
			<td>Molecular Probes</td>
			<td>A22287</td>
			<td>One to one fifty ratio</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>

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

	...

Take flow cytometry tubes containing unloaded Raji cells and Staphylococcus aureus enterotoxin B or SEB-loaded Raji cells.
These SEB-loaded Raji cells function as surrogate antigen-presenting B cells containing the superantigen SEB complexed to class II MHC molecules.
Incubate with pan-leukocytes.
The T cell receptors, TCRs on T cells bind to the SEBs on Raji cells, which, along with co-stimulatory signals, establish a stable cell-cell junction, an immune synapse, leading to T cell activation.
This triggers intracellular signaling pathways, causing the actin cytoskeleton rearrangement, with the clustering of signaling molecules and surface receptors within the synapse.
Post-incubation, fix the cells to preserve their cellular structures. Permeabilize the cells to access intracellular targets.
Add fluorophore-labeled anti-CD3 antibodies, phalloidin, and DAPI, which bind to CD3 in the TCR complex, actin filaments, and DNA, respectively.
Using an imaging flow cytometer, visualize fluorescent signals from CD3 and actin to determine their enrichment at the synapse in the presence of SEB.&#160;

29 조회수. 이미징 유세포 분석을 사용한 인간 T 세포와 SEB 탑재 라지 세포 간의 면역 시냅스 분석

비디오: 이미징 유세포 분석을 사용한 인간 T 세포와 SEB 탑재 라지 세포 간의 면역 시냅스 분석 - 실험

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.

유리 - 지원 평면 지질 이중층에 자연 살해 세포 면역 시냅스의 슈퍼 해상도 이미징

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 ...

연구

JoVE Journal

면역학 및 감염병학

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'.

이미징 면역 시냅스 역학에 대한 내피 평면 셀 모델

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

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

T 세포 면역 시냅스의 미립자 출력을 연구하기 위해 비드 지원 지질 이중층의 준비

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

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

내레이션 대본

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