real-time analysis of resident t cell migration in different tumor regions using confocal microscopy

Real-Time Analysis of Resident T Cell Migration in Different Tumor Regions Using Confocal Microscopy

Dilute the required fluorescently labeled antibodies and the fluorescence nuclear dye, DAPI, to the required final concentrations in phenol red-free RPMI medium. Next, remove the culture plate from the incubator and using a fine tip, aspirate out the liquid inside the stainless steel washers.
Now, without touching the slice, add 40 microliters of the diluted antibodies onto each tumor slice. To allow for staining, incubate the plate at 37 degrees Celsius for 15 minutes. After the staining step is complete, using fine forceps, remove the washers. Then, gently take out the slices and dip them in phenol red-free RPMI1640 medium for 10 seconds. Place the slices back onto the cell culture inserts and add a drop of phenol red-free RPMI1640 medium over each slice. Incubate the plate at 37 degrees Celsius for 10 minutes and then proceed to imaging.
Several hours prior to starting this experiment, set the temperature of the microscope heat chamber to 37 degrees Celsius, then, prepare the microscope set up to constantly perfuse tumor slices with oxygenated phenol red-free RPMI medium, and to aspirate the solution to a waste collection flask using a peristaltic pump.
With the help of fine forceps, lift the stained slice out of the six-well plate, and transfer it to a 35-millimeter plastic dish filled with phenol red-free RPMI medium, then, place a slice anchor over the slice to support it. Next, place this Petri dish on the imaging stage of the microscope.
After connecting the inlet and outlet tips of the perfusion system, turn on the peristaltic pump to allow the oxygenated medium to go through the perfusion tubes. Lower the water immersion objective to the slice, and focus on the top of the slice with bright-field light or with the appropriate wavelength.
Then, using appropriate filters, visualize and select a region of interest that contains all the fluorescently labeled cells of interest namely the CD8 T cells, the tumor islets, and the CD90-positive stromal cells. Set up an imaging session using appropriate laser intensity and exposure times to suit the brightness of the fluorescent signals observed. Then, set up the Z-stack thickness to image within the tumor slice.
Next, select the Z-stack image time interval and the total recording time to capture images at specific time intervals. After capturing and exporting the fluorescent images, analyze CD8 T cell migration as described in the text protocol.

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Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

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1. Immunostaining of resident CD8 T cells and tumor cells
<ol>
	<li>Dilute the antibodies (final concentration 10 &#181;g/mL) and the nuclear dye (final concentration 1 &#181;g/mL) in RPMI-1640 media without phenol red. 
	NOTE: Use fluorescently-coupled anti-CD8 (murine IgG, clone SK1) and anti-EpCAM (epithelial cell adhesion molecule) (murine IgG HEA-125) antibodies to label CD8 T lymphocytes and tumor epithelial cells, respectively. Use fluorescently-coupled anti-CD90/Thy1 (murine IgG, clone 5E10) antibody to label fibroblasts and activated endothelial cells. Use 4&#39;,6-diamidino-2-phenylindole (DAPI) to label the nucleus of cells with a compromised plasma membrane in order to assess tissue viability.</li>
	<li>Remove the culture plates from the incubator and use a pipette to aspirate any liquid inside the stainless steel washers. Do not remove the 1.1 mL RPMI-1640 media placed under the cell culture inserts.</li>
	<li>Without touching the slice and using a pipette tip, add 40 &#181;L of the solution containing the antibodies onto each tumor slice.</li>
	<li>Incubate the plate at 37 &#176;C for 15 min to allow antibody staining.</li>
	<li>With fine forceps, remove the washers, take out the slices, and dip them 10 s in RPMI-1640 media without phenol red. Place the slices back onto the cell culture inserts and add a drop of phenol red-free RPMI-1640 medium on each individual slice. Maintain the plate at 37 &#176;C for 10 min. Proceed to imaging.</li>
</ol>
2. Imaging and analyzing resident CD8 T cell migration within human lung tumor slices
NOTE: The confocal microscope used in this protocol is an upright spinning disk equipped with a 25x water immersion objective (25x/0.95 N.A.).
<ol>
	<li>Preparing microscope setup
	<ol>
		<li>Set the temperature of the microscope heat-chamber at 37 &#176;C a few hours before starting the imaging session.</li>
		<li>Set up a system to constantly perfuse tumor slices with oxygenated (5% CO2, 95% O2) phenol red-free RPMI medium (Figure 1). Use a peristaltic pump to flow the perfusion media into the imaging chamber and aspirate the solution to a waste collection flask.</li>
		<li>Run the peristaltic pump to allow the oxygenated solution to go through the perfusion tubes.</li>
	</ol>
	</li>
	<li>With fine forceps, take out the slice from the 6-well plate and transfer it to the 35-mm plastic dish filled with phenol red-free RPMI medium. Immobilize the slice with a 19.7 mm diameter slice anchor with 2 mm spaced threads. Place the Petri dish on the imaging stage of the microscope. Connect the inlet and outlet tips of the perfusion system. Turn on the perfusion system and set the media flow rate to 0.8 mL/min.</li>
	<li>Lower the water-immersion objective to the slice. Focus on the top of the slice with bright field light. Using appropriate fluorescent light, select a region of interest that contains CD8 T cells, tumor islets (positive for EpCAM), and a stroma area (positive for CD90). Check the viability of cells within the slice by assessing DAPI staining at the cut surface and deeper into the tissue. 
	NOTE: A healthy slice contains only a minority of DAPI-positive cells at several microns from the cut surface.</li>
	<li>Set an imaging session with the following actions.
	<ol>
		<li>Depending on the intensity of the fluorescent signal of the 3 fluorophores, set the exposure time between 50 to 800 ms and the laser intensity between 20 to 40%.</li>
		<li>Select the z stack thickness to image within the tumor slice. 
		NOTE: Here, 10-12 optical planes spanning a total depth of 60-70 &#181;m in the z dimension were captured.</li>
		<li>Select the start position at approximately 10 &#181;m below the first labeled CD8 T cells.</li>
		<li>Define the time interval between each z-stack image between 10 to 30 s and the total recording time between 10 to 30 min.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Upright confocal microscope</td>
			<td>Leica</td>
			<td>The use of a spinning disk confocal is recommended</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>This sofware is used for visualization of images and tracking of T cell migration</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>World Precision Instruments</td>
			<td>14142</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm Culture inserts</td>
			<td>Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) medium, GlutaMAX Supplement</td>
			<td>ThermoFisher</td>
			<td>61870010</td>
			<td>Complete RPMI-medium is made by adding 10 % heat-inactivated fetal calf serum and Penicillin/ streptomycin</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>ThermoFisher</td>
			<td>20012019</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Invitrogen</td>
			<td>14170088</td>
			<td></td>
		</tr>
		<tr>
			<td>Low gelling temperature Agarose, type VII-A</td>
			<td>Sigma-Aldrich</td>
			<td>A0701</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-toxic butyl Cyanoacrylate Glue, Vetbond</td>
			<td>3M</td>
			<td>1469</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice anchors, 19.7 diameter, 2 mm spacing threads</td>
			<td>Warner Instruments</td>
			<td>64-1415</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel washers, 5 mm of inner diameter</td>
			<td>Amazon</td>
			<td>B004K1FDGQ</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605-conjugated anti-CD8 (clone SK1)</td>
			<td>BD Biosciences</td>
			<td>565289</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-EpCAM (clone HEA-125)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-113</td>
			<td></td>
		</tr>
		<tr>
			<td>BV510-conjugated anti-CD90 (clone 5E10)</td>
			<td>Biolegend</td>
			<td>328125</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time analysis of resident t-cell migration in different tumor regions using confocal microscopy

Source: Peranzoni, Elisa, et al. <a href="https://app.jove.com/v/55709">Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy.</a> J. Vis. Exp. (2017).
This video describes the imaging of resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

<img alt="Figure 1" src="/files/ftp_upload/22069/22069_Figure1.jpg" /> 
Figure 1: Photographs of the perfusion system. A) The phenol red-free RPMI solution is enriched in 5% CO2, 95% O2. B) The solution is then perfused by a peristaltic pump. C) The RPMI solution enters the culture dish via the inlet. At the outlet, the solution is aspirated by the pump from the chamber into a waste container. Note that the aspiration tip is set at a level to de...

1. Immunostaining of resident CD8 T cells and tumor cells

	Dilute the antibodies (final concentration 10 &#181;g/mL) and the nuclear dye (final ...

Begin with a lung tumor slice containing resident T cells.
T cells migrate through the tumor microenvironment and interact with tumor cells and stromal elements, performing immune functions.
The composition of the tumor microenvironment strongly influences T cell displacement and velocity.
Add distinct fluorescently-coupled antibodies to label T cells, tumor cells, and stromal elements in the microenvironment.
Add DAPI, which infiltrates cells with a damaged plasma membrane to stain the nucleus, enabling the visualization of necrotic regions within the tumor.
Observe the stained sample under a confocal microscope.
Using a suitable fluorescence light, select a tumor region of interest containing resident T cells. Evaluate cell viability by assessing DAPI-positive cells.
For real-time analysis, define time intervals and total recording time.
To capture z-stack images, start at a depth below labeled T cells and capture images at various time intervals.
Utilize tracking software to compare the T cell displacement length and average velocity in different tumor regions.

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Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

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Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

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Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

Real-Time Analysis of Resident T-cell Migration in Different Tumor Regions Using Confocal Microscopy

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Real-Time Analysis of Resident T Cell Migration in Different Tumor Regions Using Confocal Microscopy