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><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 &amp;micro;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&amp;rsquo;s L-15 medium without phenol red (Gibco) supplemented with 1-5 g/L glucose</td></tr><tr><td>Liebovitz&#039;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&amp;iuml;ve T cell isolation kit</td><td>Biolegend</td><td>480043</td><td></td></tr><tr><td>Mouse E-cadherin</td><td>R&amp;amp;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 &amp;mu;M &amp;beta;-mercaptoethanol (Sigma-Aldrich)</td></tr><tr><td>Recombinant mouse ICAM-1 Fc chimera</td><td>R&amp;amp;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&amp;amp;D systems</td><td></td><td>or equivalent</td></tr><tr><td>VCAM-1 Fc chimera</td><td>R&amp;amp;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

1.3K Views.  University of Rochester. Het doel van ons onderzoek is om T-celmigratiepatronen te begrijpen in zeer gedefinieerde omgevingsomstandigheden om geïnformeerde in-vitro-experimenten mogelijk te maken en moleculaire interacties te ontcijferen die van invloed zijn op migratie. Live beeldvorming, zoals intravitale multifotonenmicroscopie, wordt gebruikt voor in vivo begrip van de kinetiek van immuuncellen. Maar microfluïdische apparaten zijn een veelgebruikt in-vitro-instrument dat nauwkeurige controle biedt over de micro...

Video: Auteur in de schijnwerpers: ziektemechanismen begrijpen door real-time analyse van T-celmigratie

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 ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cancer Research

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Bioengineering

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

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 ...

Flow Cytometry-Based Monitoring of Cancer-Immunity Cycle 

Co-Culture In Vitro Systems to Reproduce the Cancer-Immunity Cycle

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between cancer and immune cells, the so-called cancer-immunity cycle. In vitro co-culture systems combined with multiparametric flow cytometry (mFC) and tumor-on-a-chip microfluidic devices (ToCs) enable simple, fast, and reliable monitoring and characterization of each step of the cancer-immunity cycle and lead to the identification of the mechanisms responsible for tipping the balance between cancer immunosurveillance and immunoevasion. A thorough understanding of the dynamic interplays between cancer and immune cells provides critical insights to outsmart tumors and will accelerate the pace of therapeutic personalization and optimization in patients. Specifically, here we detail a straightforward mFC- and ToC-assisted protocol for unraveling the dynamic complexities of each step of the cancer-immunity cycle in murine cancer cell lines and mouse-derived immune cells and focus on immunosurveillance. Considering the time- and cost-related features of this protocol, it is certainly feasible on a large scale. Moreover, with minor variations, this protocol can be both adapted to human cancer cell lines and human peripheral-blood-derived immune cells and combined with genetic and/or pharmacologic inhibition of specific pathways in order to identify biomarkers of immune response.

Monitoring the Cancer-Immunity Cycle and Exploring Tumor Microenvironment Dynamics

Here, we detail a simple, fast, and reliable multiparametric flow cytometry- and tumor-on-a-chip-assisted protocol to monitor and characterize the steps constituting the cancer-immunity cycle. Indeed, a thorough understanding of the interplays between cancer and immune cells provides critical insights to outsmart tumors and guide clinical care.

Fundamental cancer research and the development of effective counterattack therapies both rely on experimental studies detailing the interactions between ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

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

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