Measuring Leukocyte Migration to Nucleotides

Abstract

Extracellular nucleotides are potent damage-associated molecular patterns that shape the immune response to cell stress and tissue damage. These nucleotides are sensed by purinergic receptors and mediate a wide range of cellular effects. Among the best characterized of these effects is cellular migration. While the motility responses of leukocytes to nucleotides can be achieved by microscopic live-cell imaging approaches, such systems are time-consuming and require costly equipment and analysis tools not readily available to all researchers. Transwell migration chambers are a widely used alternative to microscopy due to their relatively low cost and moderate through-put capacity. However, extracellular nucleotides are labile and rapidly degraded in serum-containing cell cultures due to the presence of phosphohydrolases. Thus, evaluating leukocyte migration to nucleotides presents a number of challenges not seen with more stable classes of chemoattractants like proteins and lipids. Here we describe a method to measure leukocyte migration to nucleotides that is cost-effective, rapid and produces robust and reproducible migration of leukocytes using transwell migration chambers.