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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

All procedures were approved by Rockefeller University's Institutional Animal Care and Use Committee. All animals were housed under specific pathogen-free conditions.

1. In Vitro Chemotaxis

1. 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.
2. In a 35 mm cell culture dish, label 3.5 x 10⁵ THP-1 cells per condition with calcein acetoxymethyl (calcein-AM; 2....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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