DifferentialMeasurements of Acidification WithinNeutrophil Swarms

Abstract Neutrophil swarming is a coordinated process triggered by interaction with targets that are significantly larger in size than the neutrophil size. While the molecular mechanisms underlying swarming are being extensively studied, the physicochemical microenvironment within swarms receives significantly less attention. Here, we probe the biochemical properties of the confined microenvironment within neutrophil swarms, focusing on pH changes during swarming. To this end, we developed a microfabricated swarming platform incorporating target particles loaded with the pH-sensitive fluorescent dye pHrodo. Using this system, we demonstrate that swarms are comprised of multiple layers of neutrophils and that significant acidification occurs within neutrophil swarms, whereas the surrounding environment remains relatively unchanged. Furthermore, we show that this acidification is dependent on neutrophil density and media buffering capacity. These findings suggest that neutrophils can actively regulate the microenvironment within swarms, providing new insights into collective neutrophil responses.