Rapid quantitation of neutrophil chemotaxis: Use of a polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly

Abstract

A neutrophil chemotaxis assay was developed which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 μm thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20–50% fall off polyvinylpyrrolidone-coated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm × 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 μl of attractant per assay well. Neutrophil chemotaxis was complete within 10–20 min at 37°C, with 20–30% of the cells migrating to N-formyl-methionyl-leucyl-phenylalanine and 40–50% migrating to complement derived C5a.