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 b...

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

<ol>
	<li>Sun, L., Su, Y., Jiao, A., Wang, X., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+in+health+and+disease.">T cells in health and disease.</a> Signal Transduct Target Ther. 8 (1), 235 (2023).</li><li>Fowell, D. J., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatio-temporal+control+of+effector+T+cell+migration.">The spatio-temporal control of effector T cell migration.</a> Nat Rev Immunol. 21 (9), 582-596 (2021).</li><li>Tabdanov, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+t+cells+to+enhance+3d+migration+through+structurally+and+mechanically+complex+tumor+microenvironments.">Engineering t cells to enhance 3d migration through structurally and mechanically complex tumor microenvironments.</a> Nat Commun. 12 (1), 2815 (2021).</li><li>Kameritsch, P., Renkawitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+leukocyte+migration+strategies.">Principles of leukocyte migration strategies.</a> Trends Cell Biol. 30 (10), 818-832 (2020).</li><li>Flaherty, S., Reynolds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+naive+CD4++T+cell+isolation+and+in+vitro+differentiation+into+t+cell+subsets.">Mouse naive CD4+ T cell isolation and in vitro differentiation into t cell subsets.</a> J Vis Exp. (98), e52739 (2015).</li><li>Sailer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pd-1(hi)+CAR-T+cells+provide+superior+protection+against+solid+tumors.">Pd-1(hi) CAR-T cells provide superior protection against solid tumors.</a> Front Immunol. 14, 1187850 (2023).</li><li>Lim, K., Hyun, Y. M., Lambert-Emo, K., Topham, D. J., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+integrin+mac-1+in+vivo.">Visualization of integrin mac-1 in vivo.</a> J Immunol Methods. 426, 120-127 (2015).</li><li>Lim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+trails+guide+influenza-specific+CD8++T+cells+in+the+airways.">Neutrophil trails guide influenza-specific CD8+ T cells in the airways.</a> Science. 349, 4352 (2015).</li><li>Lim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+neutrophil+efferocytosis+shapes+T+cell+immunity+to+influenza+infection.">In situ neutrophil efferocytosis shapes T cell immunity to influenza infection.</a> Nat Immunol. 21 (9), 1046-1057 (2020).</li><li>Reilly, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T(rm)+integrins+CD103+and+CD49a+differentially+support+adherence+and+motility+after+resolution+of+influenza+virus+infection.">T(rm) integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection.</a> Proc Natl Acad Sci U S A. 117 (22), 12306-12314 (2020).</li><li>Amitrano, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+CD8(+)+T+cell+metabolism+and+effector+functions.">Optical control of CD8(+) T cell metabolism and effector functions.</a> Front Immunol. 12, 666231 (2021).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+chemokine+receptor+signal+and+t-cell+migration.">Optogenetic control of chemokine receptor signal and t-cell migration.</a> Proc Natl Acad Sci U S A. 111 (17), 6371-6376 (2014).</li><li>Capece, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+intracellular+pool+of+IFA-1+is+critical+for+asymmetric+CD8(+)+T+cell+activation+and+differentiation.">A novel intracellular pool of IFA-1 is critical for asymmetric CD8(+) T cell activation and differentiation.</a> J Cell Biol. 216 (11), 3817-3829 (2017).</li><li>Hirai, T., Whitley, S. K., Kaplan, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+function+of+memory+CD8(+)+T+cells+in+skin.">Migration and function of memory CD8(+) T cells in skin.</a> J Invest Dermatol. 140 (4), 748-755 (2020).</li><li>Mouchacca, P., Schmitt-Verhulst, A. M., Boyer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cytolytic+T+cell+differentiation+and+granule+exocytosis+with+T+cells+from+mice+expressing+active+fluorescent+granzyme+B.">Visualization of cytolytic T cell differentiation and granule exocytosis with T cells from mice expressing active fluorescent granzyme B.</a> PLoS One. 8 (6), e67239 (2013).</li><li>Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neutrophil+chemotaxis.">Analysis of neutrophil chemotaxis.</a> Methods Mol Biol. 370, 23-36 (2007).</li><li>Heit, B., Tavener, S., Raharjo, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intracellular+signaling+hierarchy+determines+direction+of+migration+in+opposing+chemotactic+gradients.">An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients.</a> J Cell Biol. 159 (1), 91-102 (2002).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> J Cell Biol. 75 (2), 606-616 (1977).</li><li>Jowhar, D., Wright, G., Samson, P. C., Wikswo, J. P., Janetopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+microfluidic+device+for+the+study+of+cell+migration+during+chemotaxis.">Open access microfluidic device for the study of cell migration during chemotaxis.</a> Integr Biol (Camb). 2 (11-12), 648-658 (2010).</li><li>Sai, J., Walker, G., Wikswo, J., Richmond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL+sequence+in+the+LLKIL+motif+in+CXCR2+is+required+for+full+ligand-induced+activation+of+Erk,+Akt,+and+chemotaxis+in+HL60+cells.">The IL sequence in the LLKIL motif in CXCR2 is required for full ligand-induced activation of Erk, Akt, and chemotaxis in HL60 cells.</a> J Biol Chem. 281 (47), 35931-35941 (2006).</li><li>Choi, Y., Sunkara, V., Lee, Y., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhausted+mature+dendritic+cells+exhibit+a+slower+and+less+persistent+random+motility+but+retain+chemotaxis+against+ccl19.">Exhausted mature dendritic cells exhibit a slower and less persistent random motility but retain chemotaxis against ccl19.</a> Lab Chip. 22 (2), 377-386 (2022).</li><li>Harding, M. G., Zhang, K., Conly, J., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+crawling+in+capillaries:+A+novel+immune+response+to+staphylococcus+aureus.">Neutrophil crawling in capillaries: A novel immune response to staphylococcus aureus.</a> PLoS Pathog. 10 (10), e1004379 (2014).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Zanker, K. S., Dittmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+within+a+three-dimensional+collagen+matrix.">Analysis of cell migration within a three-dimensional collagen matrix.</a> J Vis Exp. (92), e51963 (2014).</li><li>Wu, P. H., Giri, A., Sun, S. X., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+migration+does+not+follow+a+random+walk.">Three-dimensional cell migration does not follow a random walk.</a> Proc Natl Acad Sci U S A. 111 (11), 3949-3954 (2014).</li><li>Lammermann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Overstreet, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+interstitial+migration+of+effector+CD4(+)+T+cells+is+dependent+on+integrin+&#945;v.">Inflammation-induced interstitial migration of effector CD4(+) T cells is dependent on integrin &#945;v.</a> Nat Immunol. 14 (9), 949-958 (2013).</li></ol>

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.

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

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

The goal of our research is to understand T cell migration patterns in highly defined environmental conditions to enable informed in vitro experimentation and decipher molecular interactions impacting migration. Live imaging, such as intravital multiphoton microscopy, is used for in vivo understanding of immune cell kinetics. But microfluidic devices are a common in vitro tool that offer precise control over the microenvironment that cannot be afforded in a live specimen.Our protocol allows us to analyze intricate signaling pathways and provides better understanding for targeted in vitro experiments to further address the biological roles of individual signals in a cell type specific manner. Our method is simple, reproducible and easy to access in research labs that have a brightfield microscope featuring an attached digital camera. Therefore, our approach ensures a reliable set of methods to study dynamic cell migration events.Our lab will focus on investigating the migration of immune cells, including T cells and neutrophils in a variety of disease settings, such as tumors and infection, thereby comprehending the mechanisms that are important to regulate immune cell migration. This will help in developing the therapeutic methods to help treat and prevent disease.

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...

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.

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