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Updated: Jun 30, 2026
Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo
Published on: October 30, 2017
A high-throughput chemotaxis assay for assessing immunotoxicity using multiple human immune cell types
Drake W Phelps1,2, Nadia Barbo1,2, Stephanie N Caty1,2
1Center for Computational Toxicology and Exposure, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, NC, US.
New approach methodologies (NAMs) for immunotoxicity are critically needed for rapidly identifying immunotoxicants, filling gaps for data-poor chemicals, and reducing the number of vertebrate animals used in toxicity testing. Current immunotoxicity testing guidelines rely on low-throughput rodent models that do not specifically assess key functions of the immune system, including chemotaxis. Alteration of chemotaxis is considered a key characteristic by which immunotoxicants may detrimentally impact the immune system. Current methods for measuring chemotaxis in a high-throughput manner are lacking. To address this, we have developed an in vitro assay to measure chemotaxis in two different immune cell types using transwell culture plates and an automated high-content imaging platform in a multi-concentration format. Our results corroborated other studies showing that Jurkat T cells and neutrophil-like HL60 (nHL60) cells respond to chemoattractants CXCL12 and fMLP, respectively, in a biphasic manner. Cytochalasin D, a specific inhibitor of actin polymerization, inhibited chemotaxis of both Jurkat cells and nHL60 cells in a concentration-dependent manner. An inhibitor of CXCR4 also decreased Jurkat chemotaxis towards CXCL12, as did cyclosporin A, a known T cell immunotoxicant. Rapamycin and cyclosporin A did not inhibit nHL60 chemotaxis. Assay performance metrics indicate that Jurkat cells and nHL60 cells may both be suitable for screening a wide variety of immunotoxicants, but more studies are needed to assess sensitivity and specificity. This work represents a NAM for identifying potential immunotoxicants that inhibit chemotaxis for future large-scale screening applications.
New approach methodologies (NAMs) for immunotoxicity are critically needed for rapidly identifying immunotoxicants, filling gaps for data-poor chemicals, and reducing the number of vertebrate animals used in toxicity testing. Current immunotoxicity testing guidelines rely on low-throughput rodent models that do not specifically assess key functions of the immune system, including chemotaxis. Alteration of chemotaxis is considered a key characteristic by which immunotoxicants may detrimentally impact the immune system. Current methods for measuring chemotaxis in a high-throughput manner are lacking. To address this, we have developed an in vitro assay to measure chemotaxis in two different immune cell types using transwell culture plates and an automated high-content imaging platform in a multi-concentration format. Our results corroborated other studies showing that Jurkat T cells and neutrophil-like HL60 (nHL60) cells respond to chemoattractants CXCL12 and fMLP, respectively, in a biphasic manner. Cytochalasin D, a specific inhibitor of actin polymerization, inhibited chemotaxis of both Jurkat cells and nHL60 cells in a concentration-dependent manner. An inhibitor of CXCR4 also decreased Jurkat chemotaxis towards CXCL12, as did cyclosporin A, a known T cell immunotoxicant. Rapamycin and cyclosporin A did not inhibit nHL60 chemotaxis. Assay performance metrics indicate that Jurkat cells and nHL60 cells may both be suitable for screening a wide variety of immunotoxicants, but more studies are needed to assess sensitivity and specificity. This work represents a NAM for identifying potential immunotoxicants that inhibit chemotaxis for future large-scale screening applications.
