A Novel Image‐Based Approach for Analyzing Neutrophil Chemotaxis Using a Boyden Chamber Assay

Chemotaxis is a fundamental biological process in which cells such as leukocytes migrate directionally from one site to another in response to a chemical gradient. This process of cellular migration is critical for execution of appropriate physiological responses to inflammation, as observed during immune defense and wound healing responses. Neutrophils being the first responders to the sites of infection or injury are one such type of cells. A neutrophil chemotaxis assay is a commonly used laboratory technique used to measure the directed movement of neutrophils toward a chemical signal termed a chemoattractant. It is used in immunology and inflammation research to study directed immune cell migration and to assess the effects of various molecules on cell migration. When studying neutrophil chemotaxis, current methods rely on counts of cells that have migrated across a membrane or substrate and observation of the migrated cells under a microscope. While useful, this approach fails to account for cells that have been activated but remain adherent to the substrate or cells that are in the intermediate stages of transmigration. Such limitations restrict the scope of information obtained and may lead to incomplete interpretations of neutrophil chemotaxis. To address this, we developed a new image analysis method that directly addresses the membrane in a chemotaxis chamber called the Boyden chamber. This improved technique allows quantification of neutrophils that have migrated as well as those that are adherent or actively transmigrating. By including all three stages of neutrophil movement, it provides a more comprehensive and physiologically relevant understanding of chemotaxis. It offers researchers a powerful tool to dissect the dynamics of immune cell behavior with greater sensitivity and accuracy, paving the way for deeper insights into immune responses and potential therapeutic interventions in inflammatory diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Assay for the migration of bone marrow-derived neutrophils using the Boyden chamber Basic Protocol 2: Analysis of adherent, transmigrating, and transmigrated cells.