Name: quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo
Uploaded: 2017-10-30T13:00:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

This work was supported by the Bernard L. Schwartz Program for Physician Scientists at The Rockefeller University, the Robertson Therapeutic Development Fund at The Rockefeller University, and the Sackler Center for Biomedicine and Nutrition Research at The Rockefeller University.

All procedures were approved by Rockefeller University&#39;s Institutional Animal Care and Use Committee. All animals were housed under specific pathogen-free conditions.
1. In Vitro Chemotaxis
<ol>
	<li>Culture THP-1 cells6 in Roswell Park Memorial Institute 1640 (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 25 mM) (complete media), maintaining the density between 0.5-1.0 x 106 cells/mL.</li>
	<li>In a 35 mm cell culture dish, label 3.5 x 105 THP-1 cells per condition with calcein acetoxymethyl (calcein-AM; 2.5 &#181;M) or other suitable fluorescent dye in complete media at 37 &#176;C for 30 min. 
	NOTE: Dyes must be cell membrane-permeable and compatible with live-cell labeling.</li>
	<li>Pre-warm a plate reader to 37 &#176;C. 
	NOTE: In addition to temperature control, bottom-reading functionality is also required. See step 1.10.2 for an alternative to a plate reader for quantification of migrating cells.</li>
	<li>While cells are labeling, prepare the assay plate and lower chamber.
	<ol>
		<li>Use a 24-well tissue culture plate, with one condition per well. Perform each condition in triplicate.</li>
		<li>Add 750 &#181;L of Hank&#39;s Buffered Salt Solution (HBSS) buffered with 25 mM HEPES and 0.1 % bovine serum albumin (BSA) to the 24-well plate. This serves as the basal control condition.
		<ol>
			<li>Use other wells for comparator conditions, always maintaining the total volume in the bottom chamber at 750 &#181;L. 
			NOTE: In this example, human monocyte chemoattractant protein-1 (MCP-1) serves as the positive control for chemotaxis7.
			<ol>
				<li>Assemble the reagent mixture by combining HBSS with 25 mM HEPES, BSA (0.1 %), and MCP-1 (75 ng/mL). Use adult bovine serum (2.7 % v / v) with MCP-1 as an additional positive control. 
				NOTE: Chemokine and chemokine concentration will depend on the chemotactic axis under study. Lyophilized MCP-1 is resuspended in distilled water with BSA (0.1%) to make a stock solution with a final concentration of 50 &#956;g/mL. This can be diluted in water to a working solution of 10 &#956;g/mL. Add 5.6 &#956;L of this solution to the lower chamber.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>After the composition of the bottom chambers is complete, place a porous insert into each well, ensuring that the tabs of the insert align with the notches of the plate. 
		NOTE: Choose the appropriate pore size for the inserts. For monocytes and lymphocytes, a pore size of 3 &#956;m performs well. Some cell types and/or chemokine systems may require a matrix-coated surface for optimal migration. For example, for measuring THP-1 monocyte chemotaxis towards MCP-1, fibronectin or collagen-coated inserts are recommended. For plate reader for quantitation, the insert must have a light-impermeant coating, such as polyethylene terephthalate, to prevent detection of non-migrating cells in the upper chamber8.</li>
	</ol>
	</li>
	<li>After incubating cells for 30 min with calcein-AM, transfer cell suspension to a 15mL conical tube. Centrifuge cells at 1,000 x g for 2 min. Visually inspect cells to confirm uptake of calcein-AM (Figure 1).</li>
	<li>Carefully remove the supernatant using a vacuum aspirator. Wash labeled cells in serum-free RPMI 1640 supplemented with HEPES 25 mM. Centrifuge cells at 1,000 x g for 2 min and discard the supernatant.</li>
	<li>Resuspend cells in serum-free RPMI 1640 supplemented with HEPES 25 mM. Count cells and adjust the volume so that the cells are at a final concentration of 1.0-1.5 x 106 cells/mL.</li>
	<li>Dispense 250 &#956;L of the cell suspension into the insert (upper chamber).</li>
	<li>After all upper chambers have been filled, gently lift the plate, and inspect the underside for the presence of air bubbles that may interfere with migration and detection. To remove a bubble, first try removing then re-positioning the insert. If unsuccessful, use a 27 G needle to puncture the bubble and then re-position the insert.</li>
	<li>Place the plate into the pre-warmed 37 &#176;C plate reader.
	<ol>
		<li>Acquire fluorescence readings from the bottom at 1-3 min intervals. When using calcein-AM, set the fluorescence excitation and emission wavelengths at 485 nm and 520 nm, respectively. Depending on the cell type and chemokine system, chemotaxis typically occurs over 1-4 h (Figure 2A).</li>
		<li>As an alternative to continuous measurements using a plate-reader, image the wells using an inverted fluorescent microscope (Figure 2B) capable of excitation and detection of light at 485 nm and 520 nm, respectively. Use a low magnification to capture the majority of the underside of the insert. Quantify cells by processing the images with ImageJ or similar software9.</li>
	</ol>
	</li>
</ol>
2. Adoptive Transfer of Murine Lymphocytes
<ol>
	<li>Preparation of donor cells
	<ol>
		<li>Anesthetize 8-12 week-old C57BL/6J male donor mice with isoflurane anesthesia (1-3 %) using a vaporizer. Confirm appropriate depth of anesthesia by toe-pinch.</li>
		<li>Place the mouse in the supine position. Make a midline incision with a scalpel and locate the spleen in the upper left peritoneal space. Excise the whole spleen and place into complete media (RPMI supplemented with 10% FBS and 25 mM HEPES; 1 mL/spleen) on ice. Euthanize anesthetized donor animals by decapitation.</li>
		<li>Grind spleen tissue gently through 40 &#181;m nylon mesh into complete media (1 mL /spleen) using the end of a syringe plunger to make a single-cell suspension and transfer to a 15 mL conical tube.</li>
		<li>Add 4 volumes of ammonium chloride potassium (ACK) lysis buffer and incubate for 5 min at room temperature to lyse erythrocytes.</li>
		<li>Centrifuge the cells at 1,000 x g for 2 min at room temperature and discard supernatant. If erythrocytes remain in the pellet, resuspend in ACK lysis buffer, incubate for 5 min at room temperature, centrifuge cells at 1,000 x g for 2 min and discard supernatant.</li>
		<li>Resuspend the cells in complete media at a concentration of 2-5x107 cells/mL, then strain the cell suspension through a 40 &#181;m nylon mesh to remove cell debris.</li>
		<li>Label 1 x 107 cells/recipient mouse with a fluorescent dye compatible with live cells and that will be retained upon subsequent fixation, such as 5-chloromethylfluorescein diacetate (final concentration 2 &#956;M)10. Incubate at 37 &#176;C for 30 min. 
		NOTE: To track more than one cell population, use other fluorescent dyes with additional cell populations. For example, a portion of donor cells can be labeled with 5-(and-6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine (final concentration 2 &#956;M)11.</li>
		<li>Add 4 volumes HBSS supplemented with 25 mM HEPES, then centrifuge at 1,000 x g for 2 min and discard supernatant. Resuspend pellet in HBSS supplemented with HEPES 25 mM to a final concentration of 1 x 108 cells/mL.
		<ol>
			<li>If using more than one fluorescent dye, keep cell populations separated until immediately prior to injection.</li>
			<li>Transfer a small aliquot (10 &#956;L) of cells of each color to a fixative solution (1 mL) for subsequent flow cytometry compensation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfer of lymphocytes
	<ol>
		<li>Anesthetize 8-12 week-old C57BL/6J male recipient mice with isoflurane (1-3 %) using a vaporizer; confirm depth of anesthesia by toe-pinch. Apply veterinary ointment to animal&#39;s eyes to prevent drying.</li>
		<li>Briefly vortex donor cells (1 s) and transfer to 1 mL injection syringe with 27 G, 0.5 in needle, combining differentially labeled cell populations, if applicable.
		<ol>
			<li>Transfer a small aliquot (10 &#956;L) of mixed cells to a fixative solution (1 mL) for subsequent flow cytometry analyses.</li>
		</ol>
		</li>
		<li>Place donor animals in the prone position. Apply mild caudal pressure to the skin dorsal to the eye, causing the eyeball to protrude slightly.</li>
		<li>Direct the needle medially and inject 100 &#181;L of donor cell suspension retro-orbitally into each recipient mouse. Cells can also be injected via the tail vein.</li>
		<li>Gradually withdraw the injection needle and place mouse alone in a recovery cage. Monitor animal until it has regained consciousness; once fully recovered return the animal to social housing.</li>
	</ol>
	</li>
	<li>Collection and Analysis
	<ol>
		<li>After 1 h, anesthetize recipient mice with isoflurane anesthesia (1-3%) using a vaporizer; confirm depth of anesthesia by toe-pinch. Harvest the spleen (or other tissue of interest) (step 2.1.2.) from each recipient mouse and place into complete media (1 mL/recipient). Euthanize anesthetized animals by decapitation. 
		NOTE: Longer intervals prior to tissue harvest will increase tissue accumulation through at least 24 h.</li>
		<li>Grind spleen tissue gently through 40 &#181;m nylon mesh into complete media using the end of a syringe plunger to make a single-cell suspension and transfer to 15 mL conical tube.</li>
		<li>Add 4 volumes of ACK lysis buffer and incubate 5 min at room temperature. Centrifuge the cell suspension at 1,000 x g for 2 min at room temperature and discard supernatant.</li>
		<li>Resuspend cells in staining buffer (phosphate-buffered saline supplemented with 1% FBS). Count cells and adjust to a final concentration of 3 x 10 7 cells/mL. Transfer 100 &#181;L of cell suspension to a 96-well round bottom plate.</li>
		<li>Stain cells for desired markers (see the Table of Materials) for analysis by flow cytometry. Add fluorophore-conjugated antibodies (see the Table of Materials) against desired markers and incubate at 4 &#176;C for 20 min in the dark. Add 100 &#181;L staining buffer, centrifuge cells at 1,000 x g for 3 min and discard the supernatant.</li>
		<li>Resuspend the pellet in 200 &#181;L staining buffer, centrifuge cells at 1,000 x g for 3 min and discard the supernatant.</li>
		<li>Resuspend the pellet in 125 &#181;L staining buffer and acquire samples using a flow cytometer using standard procedures. 
		NOTE: The green dye is excited by a 488 nm laser and the emission is detected with 505 and 530/30 dichroic filters. The orange dye is excited by a 561 nm and the emission is detected by a 582/15 dichroic filter.
		<ol>
			<li>Use saved cells from 2.1.8.2 to compensate for each fluorescent dye. 
			NOTE: Using bead-based compensation with fluorophores matching the excitation and emission spectra of the labeling dyes will result in suboptimal compensation.</li>
			<li>Use the saved input cells from step 2.2.2.1 to accurately determine the ratio of differentially labeled input cells if using more than one labeled population (Figure 3). Calculate tissue specific migration based on the ratio of labeled to unlabeled cells (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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	<li>Griffith, J. W., Sokol, C. L., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+chemokine+receptors:+positioning+cells+for+host+defense+and+immunity.">Chemokines and chemokine receptors: positioning cells for host defense and immunity.</a> Annu Rev Immunol. 32, 659-702 (2014).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76 (2), 301-314 (1994).</li><li>Ponda, M. P., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+stimulation+of+CCR7+chemotaxis+due+to+coagulation+factor+XIIa-dependent+production+of+high-molecular-weight+kininogen+domain+5.">Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.</a> Proc Natl Acad Sci U S A. 113 (45), E7059-E7068 (2016).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115, 453-466 (1962).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods Mol Biol. 294, 15-22 (2005).</li><li>Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>Leonard, E. J., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocyte+chemoattractant+protein-1+(MCP-1).">Human monocyte chemoattractant protein-1 (MCP-1).</a> Immunol Today. 11 (3), 97-101 (1990).</li><li>Crouch, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+fluorescence+based+assay+of+neurite+formation.">An automated fluorescence based assay of neurite formation.</a> J Neurosci Methods. 104 (1), 87-91 (2000).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Poot, M., Kavanagh, T. J., Kang, H. C., Haugland, R. P., Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+cell+cycle-dependent+changes+in+cell+thiol+level+by+combining+a+new+laser+dye+with+Hoechst+33342.">Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.</a> Cytometry. 12 (2), 184-187 (1991).</li><li>West, C. A., He, C., Su, M., Swanson, S. J., Mentzer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+fixation+of+thiol-reactive+fluorescent+cytoplasmic+probes+for+tracking+cell+migration.">Aldehyde fixation of thiol-reactive fluorescent cytoplasmic probes for tracking cell migration.</a> J Histochem Cytochem. 49 (4), 511-518 (2001).</li><li>Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+greater+chemotactic+network+for+lymphocyte+trafficking:+chemokines+and+beyond.">The greater chemotactic network for lymphocyte trafficking: chemokines and beyond.</a> Curr Opin Hematol. 12 (4), 298-304 (2005).</li></ol>

The authors have nothing to disclose

Quantification of immune cell migration can be accomplished using simple and rapid assays both in vitro and in vivo. We demonstrate the in vitro chemotaxis of human monocytes in response to a MCP-1 gradient and augmentation by serum. In vivo, donor murine splenocytes were differentially labeled and following adoptive transfer, were recovered from recipient animals.
Using a plate reader has the advantage of sampling several time points (as frequently as every...

Robust immunity requires the complex temporal and spatial coordination of a myriad of cell types in order to respond appropriately to injury, infection and generate self-tolerance. Several dozen chemokine receptors and their corresponding ligands have been discovered and characterized providing molecular mechanisms by which specific cells can be directed into a specific tissue at a specific time. Thus, studying chemotaxis and migration is an indispensable component of immunology research. Indeed, the described in vitro assay was recently used as a screening tool to identify a chemotactic cofactor that accelerates chemotaxis of T-cells toward C-C chemokines 19 and 213. The purpose of the methods described here are to permit quantitative assessments of immune cell chemotaxis in vitro and in vivo.
The Boyden (cell migration and invasion) chamber assay is an inexpensive, reproducible, and rapid method for assessing cell migration4,5. In the standard assay, the upper chamber is seeded with cells, and is separated by a porous insert from a lower chamber, into which the cells migrate. At the desired time, cells that have migrated to the underside of the insert can be fixed and stained for quantitation by light microscopy. However, such measurements constrain data collection to a single end point, which precludes dynamic data collection and can require extensive optimization to determine the optimal time point for analysis. Here, several adaptations are described that permit real-time, quantitative and multiplexed measurements of chemotaxis in vitro.
For in vivo studies, a functional end point is used, namely the specific accumulation of cells in a given tissue compartment. Pre-labeled donor cells are introduced into recipient animals through adoptive transfer. These donor cells can subsequently be identified by flow cytometry after recipient tissue harvest. Also presented is a co-labeling strategy that allows for the determination of trafficking of different cell types within a single recipient animal. This method eliminates the inter-animal variation from cell injection, and accounts for physiologic inter-animal variability.

<table><tbody><tr><td>RPMI-1640</td><td>Thermo Fisher Scientific</td><td>11875-093</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>15630-080</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Cell culture flask</td><td>Corning</td><td>353136</td><td></td></tr><tr><td>Calcein AM</td><td>Thermo Fisher Scientific</td><td>C1430</td><td>Excitation: 485nm, Emission: 520nm</td></tr><tr><td>Cell culture dish</td><td>Corning</td><td>1007</td><td></td></tr><tr><td>24 well plate</td><td>Corning</td><td>353504</td><td></td></tr><tr><td>Fluoroblok Fibronectin Insert</td><td>Corning</td><td>CB354597</td><td></td></tr><tr><td>15 mL conical tube</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Cell Signaling Technology</td><td>9998S</td><td></td></tr><tr><td>Human Recombinant MCP-1</td><td>Peprotech</td><td>300-04</td><td></td></tr><tr><td>Adult bovine Serum</td><td>Sigma</td><td>B9433</td><td></td></tr><tr><td>HBSS with calcium and magnesium; no phenol red</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>SpectraMax M2e plate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Olympus IX71 inverted fluorescence microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Isothesia</td><td>Henry Schein animal health</td><td>11695-6776-2</td><td>Isofluorane anesthesia</td></tr><tr><td>Cell strainer 40um nylon</td><td>Falcon Corning</td><td>352340</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>CellTracker Orange CMTMR Dye</td><td>Thermo Fisher Scientific</td><td>C2927</td><td>Excitation: 541nm, Emission: 565nm</td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Thermo Fisher Scientific</td><td>C2925</td><td>Excitation: 485nm, Emission: 520nm</td></tr><tr><td>5ml syringe</td><td>BD Syringe</td><td>309646</td><td></td></tr><tr><td>1ml TB syringe</td><td>BD Syringe</td><td>309625</td><td></td></tr><tr><td>THP-1 cell line</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Brilliant Violet 421 anti-mouse CD3 Antibody</td><td>Biolegend</td><td>100228</td><td>Excitation: 405nm, Emission: 421nm</td></tr><tr><td>Brilliant Violet 605 anti-mouse CD19 Antibody</td><td>Biolegend</td><td>115540</td><td>Excitation: 405nm, Emission: 603nm</td></tr><tr><td>96-well round bottom plate</td><td>Corning</td><td>353077</td><td></td></tr><tr><td>LSRII</td><td>BD Biosciences</td><td></td><td>Flow cytometer</td></tr></tbody></table>

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.

When using calcein-AM dye, visual inspection of cells will confirm label uptake (Figure 1). Automated fluorescent readings will track migration as cells transit onto the underside of the insert over time. These data clearly show an induction of cell migration towards MCP-1, as well as an augmentation of this response by serum (Figure 2A). Depending on the strength of the migratory stimulus, there is a lag of at least 15 min prior...

Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

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.

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 ...

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Immunology and Infection

7.1K Views.  The Rockefeller University. 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.

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

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 ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

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 ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Medicine

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

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 ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

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

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Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

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

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors