We thank previous and current members of the Kim Lab who have contributed to the development of these protocols over time. Representative data were made possible by P01 AI102851/AI/NIAID NIH HHS/United States and P01 HL018208/HL/NHLBI NIH HHS/United States. This publication was made possible in part by Grant Number T32 GM135134 from the Institutional Ruth L. Kirschstein National Research Service Award.

The animal protocols were approved by the University Committee on Animal Resources at the University of Rochester. The mice in this study were maintained in the pathogen-free space of the University of Rochester animal facility. Male/female C57BL/6 mice, aged 6-12 weeks (15-30 g), were used for the present study. Mouse tissue isolation can be performed on a benchtop with gloves to cover hands&#160;and&#160;a facemask to cover the nose and mouth, or inside a biosafety cabinet. All cell culture and plate preparation must be performed in a biosafety cabinet. The reagents and equipment used in this study are listed in the Table of Materials.
1. CD8+ T cell purification and activation
<ol>
	<li>Preparation of materials
	<ol>
		<li>For the negative selection of CD8+ T cells: Prepare a negative selection medium from the supernatant of hybridoma cell lines GK1.5 and M5/114 antibodies, which produce anti-CD4 and anti-MHCII, respectively. Mix the supernatant at a 1:1 ratio, filter it, and store it at 4 &#176;C for future use (&#62;0.5 &#181;g of each antibody/million total cells). Alternatively, commercial CD8+ T cell isolation kits are available.</li>
		<li>For activation of T cells: Add 500 &#181;L of CD3 (10 &#181;g/mL) and CD28 (16 &#181;g/mL) antibodies in 1x DPBS (without calcium and magnesium) to a non-tissue culture (TC) treated 24-well plate and store overnight at 4&#160;&#176;C to ensure plate binding. 
		NOTE: Antibodies and proteins coat non-TC-treated culture plates/dishes better than regular TC-treated ones, so non-TC-treated culture plates are used for T cell generation.</li>
	</ol>
	</li>
	<li>Prepare a 10 cm culture dish (or equivalent) by placing a 70 &#181;m cell strainer into the dish and dispense 2 mL of R9 medium into the filter (R9 medium: RPMI 1640x&#160;supplemented with 10% FBS, 1% antibiotic-antimycotic, 20 mM HEPES buffer, 1% MEM non-essential amino acids, 50 &#181;M &#946;-mercaptoethanol).</li>
	<li>Obtain a mouse with appropriate genetic background, sex, age, and weight as determined by the experimental design.</li>
	<li>Euthanize the mouse using CO2 followed by cervical dislocation, or appropriate euthanasia strategies as defined by local animal care and use protocols and approved by the institution.</li>
	<li>Place the mouse in a supine posture, stretch all four limbs perpendicular to the body, and pin footpads to a dissection board using mouse dissection T pins, or equivalent. Make a superficial, central incision through the cutaneous layer on the ventral lower abdomen with mouse surgical dissection scissors and cut straight up to the chin (~8 cm). Separate the skin from the peritoneal lining to expose lymph nodes. Stretch the skin perpendicularly away from the trunk and pin it down5.
	<ol>
		<li>Gently excise the cervical, axial, brachial, and inguinal lymph nodes bilaterally using blunt forceps (or the preferred type from a standard mouse surgical dissection kit). Transfer to the cell strainer prepared in step 1.2.</li>
		<li>Open the peritoneum and remove the spleen. Transfer to the cell strainer prepared in step 1.2.</li>
		<li>Dispose of the mouse carcass in the appropriate biohazard receptacle.</li>
	</ol>
	</li>
	<li>Prepare a single-cell suspension of the secondary lymphoid tissues by mechanical disruption over the 70 &#181;m cell strainer with 2 mL of R9 medium from step 1.2 by twisting the tissue disruption tool one-half turn clockwise and counterclockwise repeatedly. 
	NOTE: The plunger end of a luer-lock syringe, either 3 mL or 10 mL, works well to crush and homogenize the tissue. Alternative materials can be used at the user&#39;s discretion.</li>
	<li>Wash the cells, syringe end, strainer, and culture dish with 7 mL of medium, ensuring the strainer remains upright to prevent the transfer of larger pieces of tissue or cellular aggregates that may be present into the single-cell suspension.</li>
	<li>Transfer the cell suspension to a 15 mL conical tube.</li>
	<li>Pellet the cells at 270 x g&#160;at room temperature (20&#160;&#176;C) for 5 min and decant the supernatant.</li>
	<li>Add 500 &#181;L of lysing buffer for 1 min to remove red blood cells. Dilute with 9.5 mL of R9 medium and spin at 270 x g&#160;(20&#160;&#176;C) for 5 min. Decant the supernatant.</li>
	<li>Resuspend the pellet in 10 mL of the previously prepared negative selection medium containing anti-MHCII and anti-CD4 (step 1.1.1). Rock at room temperature (20&#160;&#176;C) for 30 min.</li>
	<li>Pellet the cells at 270 x g for 5 min&#160;and decant the supernatant. Wash 3x with 10 mL of R9 medium.</li>
	<li>Simultaneously, in a 15 mL conical tube, wash 200 &#181;L of sheep anti-rat IgG beads in 7 mL of medium. Place the tube on a magnet, remove the medium, and repeat the wash step twice. Resuspend the beads in 7 mL of R9 medium.</li>
	<li>Pellet the cells at 270 x g&#160;(20&#160;&#176;C) for 5 min, decant the supernatant, and then resuspend the pellet in 7 mL of bead suspension from step 1.13. Rock at room temperature (20&#160;&#176;C) for 45 min.</li>
	<li>Enrich CD8+ T cells by magnetic bead depletion. Place the conical tube directly on the magnet without a lid for 3 min to remove antibody/bead-bound cells. CD8+ cells should be retained. With the tube still in the magnet, collect the cell suspension using a serological pipet or equivalent and transfer it to a new 15 mL tube. Centrifuge at 270 x g&#160;(20&#160;&#176;C) for 5 min and wash 3x in medium.</li>
	<li>Wash the pre-coated activation plate (step 1.1.2) with 1x DPBS twice without directly pipetting on the bottom surface of the plate.</li>
	<li>Optional: Perform a live/dead lymphocyte separation to remove dead cells.
	<ol>
		<li>Transfer cells to a 15 mL conical tube. Gently add 2 mL of lymphocyte separation media to the bottom of the cell suspension. Centrifuge at 900 x g for 10 min (at room temperature) at low acceleration and deceleration. 
		NOTE: Live cells will separate at the interface of medium and separation media (middle white layer). Dead cells will pellet at the bottom of the tube and may be discarded at the end.</li>
		<li>Transfer the middle layer containing live lymphocytes to a new tube and centrifuge at 270 x g for 5 min at room temperature. Decant the supernatant.</li>
	</ol>
	</li>
	<li>Resuspend the pellet in 12 mL of medium with 10 U/mL recombinant mouse IL-2. Plate 1 mL per well in a non-TC treated 24-well plate and transfer to 37&#160;&#176;C overnight.</li>
</ol>
2. Lifting the activated CD8+ T cells
<ol>
	<li>Visualize cells using a standard benchtop light microscope, using the 4x or 10x objective. 
	NOTE: Activated T cells will appear larger and round or elongated compared to inactivated T cells and should have dense clusters of cells throughout the well.</li>
	<li>To lift the activated cells, disrupt the cells with a 1 mL pipette, making sure to mix well at the edges of the plate. Transfer cells to a new 15 mL conical tube. 
	NOTE: Activated cells are stickier and require more vigorous pipetting.</li>
	<li>Wash the wells with 1 mL of R9 medium to remove any remaining cells. Repeat once more if needed. Centrifuge the cells at 270 x g (20&#160;&#176;C) for 5 min. Discard the supernatant.</li>
	<li>Perform a live/dead lymphocyte separation to remove dead cells as in steps 1.17-1.18.</li>
	<li>Maintain T cells by live/dead cell removal every 3-4 days in R9 medium with IL-2 in a non-TC&#160;6-well plate. 
	NOTE: T cells proliferate better in wells of small dimension during early activation, possibly due to T cell-T cell contacts or T cell-derived soluble factors that help their activation. Rapid T cell proliferation causes the culture medium to yellow within 24-48 h post-culture. At this point, lift the cells and transfer them to a larger well (uncoated, non-TC 6-well plate) with sufficient fresh culture medium to feed the cells (typically 5 mL per well). Replace depleted medium with fresh whenever indicated by yellow medium color change, or every 3-4 days.</li>
</ol>
3. Preparation of glass dish
<ol>
	<li>Prepare glass cell migration chambers or plates by coating with 300 &#181;L/cm2 Protein A (0.1&#160;mg/mL) overnight at 4 &#176;C, wash twice with 1x DPBS by dispensing onto the dish and decanting before moving to the next step.</li>
	<li>Coat the migration chamber or plate with 300 &#181;L/cm2 recombinant mouse ICAM-1 or VCAM-1 Fc chimera (0.1-2.75 mg/mL) together with a recombinant mouse chemokine (0.1-10 &#181;g/mL) for 2-3 h at room temperature. Other integrin ligands such as fibronectin, mouse collagen IV, and mouse E-cadherin are rendered to directly coat the chamber at 2.5-10 &#181;g/mL overnight at 4 &#176;C.
	<ol>
		<li>Wash chambers twice with 1x DPBS by dispensing onto the dish and decanting immediately after before adding cells. 
		NOTE: T cells can migrate differently in different concentrations of integrin ligands and chemokines depending on the status of activation, differentiation, sensitization, and priming. Assaying cell migration in multiple different concentrations is strongly suggested.</li>
	</ol>
	</li>
	<li>Plate the adherent cells on glass-coated plates 24 h prior to the start of imaging if co-culturing T cells with other cells, such as cancer cells, to measure T cell killing of cancer cells in a real-time manner.</li>
</ol>
4. Preparation of cells
<ol>
	<li>Optional: Stain T cells and adherent cells with cell tracker dye of choice at 1 &#181;g/mL per 1 x 107 cells in 1x DPBS for 15 min at 37 &#176;C, or according to the manufacturer&#39;s protocol.</li>
	<li>Count cells using a hemocytometer or equivalent.</li>
	<li>Plate 1 x 105 CD8+ T cells (or desired amount) in Leibovitz&#39;s L-15 medium supplemented with 1-5 g/L glucose in a 37 &#176;C chamber. 
	NOTE: L-15 medium is formulated for use without a CO2-sodium bicarbonate system. It is buffered using free basic amino acids, phosphate buffers, and galactose to maintain pH levels.</li>
	<li>Optional: Perform integrin blocking.
	<ol>
		<li>Preincubate T cells with a blocking antibody to an integrin (1-10 &#181;g/mL, anti-mouse CD11a (M17/4), anti-mouse CD18 (M18/2), anti-mouse VLA-4 (9C10) for 10 min and allow to crawl in the presence of the same antibody.</li>
	</ol>
	</li>
</ol>
5. Time-lapse microscopy
<ol>
	<li>Perform video microscopy.</li>
	<li>For T cells, acquire brightfield and fluorescent images every 10-60 s for 10-60 min, or desired acquisition settings. 
	NOTE: T cell migration is temperature-sensitive, and the optimal temperature for mouse T cell migration is 37 &#176;C. The temperature control system that this protocol uses consists of an enclosed and insulated microscope deck with an attached heating system. The imaging chamber sits on the imaging deck. The perimeter of this chamber has a well filled with distilled water that allows consistent heat and humidity during imaging. The microwell is positioned at the center of the imaging chamber.</li>
</ol>
6. Software-assisted analysis of T cell migration
<ol>
	<li>Use the Volocity software to assess T cell migration.
	<ol>
		<li>Open Volocity and create a new image sequence.</li>
		<li>Select and move all time-lapse video microscopy movies to the blue area in Volocity.</li>
		<li>Adjust brightness and contrast using the contrast enhancement tool to the researcher&#39;s desired settings.</li>
		<li>Check image sizes: 0.325 &#181;m for 20x, 0.65 &#181;m for 10x.</li>
		<li>Sequence: set timepoints&#160;(ex: 81 images if taking an image every 15 s for 20 min, 4 per min).</li>
		<li>Uncheck all timepoints in the measurement tab.</li>
		<li>Find objects using intensity-slide the bar so it is just right of the peak.</li>
		<li>Exclude objects by size: all cells that are smaller than 10 &#181;m diameter and greater than 100 &#181;m to exclude cell debris or non-cellular particles. Exclude T cells that are migrating for less than 20% of the recording time (which can vary depending on the definition by each investigator).</li>
		<li>Ignore static objects (check box).</li>
		<li>Automatically join broken tracts (check box).</li>
		<li>Set the shortest path to no more than 20 &#181;m.</li>
		<li>The track of a cell migration is the line connecting the location of the same cell over time. Ensure that the tracking algorithm is tracking by detecting where individual objects are detected by segmentation in each frame, and the objects are matched across frames.</li>
		<li>Save a new protocol by choosing Measurements &#62; Save Protocol &#62; Name New Protocol.</li>
		<li>Click on measure all timepoints in the measurement tab.</li>
		<li>Sort tracks by timespan, high to low.</li>
		<li>Record track ID numbers for all cells with good tracks as defined by the investigator. After automatic cell tracking, manual evaluation of each cell track is necessary to exclude falsely identified or broken tracks. Exclude cells with bad tracks.</li>
		<li>Export the file as comma-separated text and transfer data to the desired analysis software. 
		NOTE: Basic parameters: Velocity (&#181;m/min), displacement (net movement, &#181;m), track length (total path length, &#181;m), and meandering index (MI; net displacement/track length. MI = 1 indicates a completely straight linear track).</li>
	</ol>
	</li>
	<li>Perform manual tracking using Image J.
	<ol>
		<li>Open plugins and select tracking and manual tracking. Set the parameters - time intervals, x/y calibration (pixel size), and z calibration (in manual tracking mode). Start individual cell tracking by clicking on add track, selecting one cell at the first time point, and continuing it through all time points. Next, click on End track. Repeat it for all the cells of interest. 
		NOTE: Trackmate, another tool for cell tracking, is an automatic tracking software available via the ImageJ plugin.</li>
	</ol>
	</li>
</ol>

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

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The authors declare that all research and manuscript preparation was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Understanding the biological impact of converging signals in vivo is challenging and not easy to interpret. The protocols presented herein provide a reasonable method to understand T cell migration in highly defined and biologically relevant conditions. These conditions can be specified based on the investigator&#39;s discretion, and the protocols can be modified to fit the needs of various T cell populations, activation status, and cell phenotype. Furthermore, many ligands and receptors can be interrogated thro...

T cells are the major effectors of the adaptive, antigen-specific immune response. On a population level, T cells are heterogeneous, comprised of cellular subsets with distinct specialized functions. Importantly, CD8+ T cells are the main cytolytic effectors of the immune system, which directly eliminate infected or dysfunctional cells1.
Mature CD8+ T cells reside in tissue and circulate through blood and lymphatics in search of antigens. During infection, T cells are presented with antigens in blood or tissue and quickly drain to the spleen or nearest draining lymph node to begin a productive immune response. In either case, T cells become activated, undergo clonal expansion, and leave the lymphatic system to enter the blood, if not already there. During this process, intracellular signaling confers the downregulation of lymphatic homing receptors and the upregulation of numerous integrin and chemokine receptors essential for tissue-specific migration2. Ultimately, the directed migration of T cells to sites of infection is driven by converging environmental signals that include integrin and chemokine signaling.
Chemokines can be broadly categorized into two main classes: (1) homeostatic signals, which are essential for differentiation, survival, and basal function, and (2) inflammatory signals, such as CXCL9, CXCL10, and CCL3, which are required for chemotaxis. Generally, chemokines create a signal gradient that drives directional migration, known as chemotaxis, in addition to activating integrin expression1. Chemotaxis is finely regulated and highly sensitive, with T cells capable of responding to tiny changes in gradient that can lead them toward a specific direction or location.
In addition to these T cell-related factors, migration is also affected by the extracellular matrix (ECM) composition and density. The ECM is made up of a dense network of proteins, including collagen and proteoglycans, which provide the scaffold for adhesive integrin receptors on T cells. Integrins are a diverse family of transmembrane proteins, each with highly specialized binding domains and downstream signaling effects. Dynamic expression of integrin receptors on the surface of a T cell enables quick adaptation to their changing environments3. Importantly, integrins connect the ECM and intracellular cytoskeletal actin networks that work together to generate the propelling force required for T cell movement.
In summary, migration patterns vary based on the immune cell phenotype or environmental signals. These complex biological processes are tightly regulated by the expression of cytokines, chemokines, and integrins on the surface of the T cell, surrounding cells, and the local, infected tissue. In vivo, these migratory mechanisms can be&#160;complex and may result from several additive signals4. Due to this complexity, it can be impossible to establish a causal relationship between seemingly interlocked variables. To overcome this, there are several in vitro approaches to study specific aspects of T cell migration such as response to specific chemokine signals and the interaction between T cell integrins and ECM binding proteins. This protocol addresses methods to isolate and activate murine CD8+ T cells, with in vitro migration assays in two-dimensional space and computational analysis tools for analyzing specified T cell migration. These methods are advantageous to the user because they do not require sophisticated materials or devices, as with some other cell migration assays described in the literature. Cell migration data generated with these methods can&#160;provide evidence of immune responses in a simplistic manner that enables further, informed investigation in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>1x DPBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td>without calcium and without magnesium</td>
		</tr>
		<tr>
			<td>6 well plate non-TC treated</td>
			<td>Corning</td>
			<td>3736</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>FisherScientific</td>
			<td>352350</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>ThermoFisher</td>
			<td>A1049201</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Allegra 6KR centrifuge</td>
			<td>ThermoScientific</td>
			<td>sorvall 16R with tx400 3655 rotor and bucket</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Beta mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>ThermoFisher</td>
			<td>C34571</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>ThermoScientific</td>
			<td>Sorvall ST 16R</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Collagen (IV)</td>
			<td>Corning</td>
			<td>354233</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>DeltaT culture dish .17 mm thick glass clear</td>
			<td>Bioptechs</td>
			<td>04200417C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Sheep anti-Rat IgG</td>
			<td>Invitrogen</td>
			<td>11035</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 15 Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12301D</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Easy sep mouse T cell isolation kit</td>
			<td>Stem Cell</td>
			<td>19851</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>SigmaAldrich</td>
			<td>F2442-500ML</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>SigmaAldrich</td>
			<td>10838039001</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td>http://fiji.sc/</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>Filter cubes</td>
			<td>Nikon or Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GK1.5</td>
			<td>ATCC</td>
			<td>TIB-207</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher</td>
			<td>15630080</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>HQ CCD camera</td>
			<td>CoolSNAP</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>http://imagej.nih.gov/ij/h</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>ImageJ automatic tracking plug in</td>
			<td></td>
			<td>http://imagej.net/TrackMate</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>ImageJ manual tracking plug in</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/track/track.html</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>L-15</td>
			<td>Various</td>
			<td>See Materials</td>
			<td>Medium Recipe: Leibovitz&#8217;s L-15 medium without phenol red (Gibco) supplemented with 1-5 g/L glucose</td>
		</tr>
		<tr>
			<td>Liebovitz&#39;s L-15 medium, no phenol red</td>
			<td>ThermoFisher</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lok disposable syringe</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Lymphocyte separation medium</td>
			<td>Corning</td>
			<td>25-072-CI</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>M5/114</td>
			<td>ATCC</td>
			<td>TIB-120</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Microscope heating system</td>
			<td>Okolab</td>
			<td>okolab.com</td>
			<td>Custom designs available</td>
		</tr>
		<tr>
			<td>Millicell EZ slide</td>
			<td>Millipore</td>
			<td>C86024</td>
			<td></td>
		</tr>
		<tr>
			<td>Mojosort mouse CD8+ Na&#239;ve T cell isolation kit</td>
			<td>Biolegend</td>
			<td>480043</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse E-cadherin</td>
			<td>R&#38;D systems</td>
			<td>8875-EC-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Mouse surgical dissection kit</td>
			<td>Fisher Scientific</td>
			<td>13-820-096</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>NIS elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>non-TC 24wp</td>
			<td>Corning</td>
			<td>353047</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Protein A</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>R9</td>
			<td>Various</td>
			<td>See Materials</td>
			<td>Medium Recipe: RPMI 1640x supplemented with 10 % FBS, 1 % antibiotic-antimycotic (Gibco), 20 mM HEPES buffer (Gibco), 1 % MEM Non-Essential Amino Acids (Gibco), 50 &#956;M &#946;-mercaptoethanol (Sigma-Aldrich)</td>
		</tr>
		<tr>
			<td>Recombinant mouse ICAM-1 Fc chimera</td>
			<td>R&#38;D systems</td>
			<td>796-IC-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL2</td>
			<td>Biolegend</td>
			<td>575410</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>RPMI 1640x</td>
			<td>ThermoFisher</td>
			<td>11875093</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>T pins</td>
			<td>Fisher Scientific</td>
			<td>S99385</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>TE2000-U microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Various recombinant mouse chemokine</td>
			<td>R&#38;D systems</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>VCAM-1 Fc chimera</td>
			<td>R&#38;D systems</td>
			<td>643-VM-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

real-time in vitro migration assay for primary murine cd8+ t cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Confirmation of T cell activation can be achieved by flow cytometry, looking for increased expression of CD69 and CD44, which are canonical markers of activation in murine T cells6. Additionally, the purity of the T cell population can be determined by flow cytometry for CD3+ CD8+ T cells. This method yields &#62;90 % CD8+ T cell population.
T cell migration can be assessed with software-assisted cell tracking programs that are both repr...

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

real-time-in-vitro-migration-assay-for-primary-murine-cd8-t-cells

Research

JoVE Journal

Immunology and Infection

1.1K Views.  University of Rochester. This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

Video: Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.