Name: quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo
Uploaded: 2017-10-30T13:00:00
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<...

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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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