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 pm 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 X 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 pl 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-formylmethionyl-leucyl-phenylalanine and 40--50% migrating to complement derived C5a.

Up-Regulation of IL-17 Is Associated with Bioactive IL-8 Expression inHelicobacter pylori-Infected Human Gastric Mucosa

“…We report that LPMC isolated from the human gastric mucosa express IL-17 at both RNA and protein levels. This appears to be consistent with the demonstration that the vast majority of lymphocytes infiltrating the gastrointestinal mucosa bear the phenotype of Ag-experienced memory T cells (20,(23)(24)(25)(26)(27)(28)(29)(30). When RNA transcripts for IL-17 were analyzed by a semiquantitative RT-PCR, an increased accumulation was found in both mucosal tissue and LPMC samples from Hp-infected patients compared with that in uninfected subjects.…”

The Mitochondrial Network of Human Neutrophils: Role in Chemotaxis, Phagocytosis, Respiratory Burst Activation, and Commitment to Apoptosis

“…Chemotaxis and random migration were evaluated with a 10-well microchamber using a modified Boyden chamber (18). Escherichia coli endotoxin-treated human serum and fMLP (10 Ϫ8 M), were used as positive chemotactic stimuli.…”

Smoke Extract Stimulates Lung Epithelial Cells to Release Neutrophil and Monocyte Chemotactic Activity

“…24 The bottom wells of the chamber were filled with 25 l of fluid containing the chemotactic stimulus or media in duplicate. A 10-mthick polyvinylpyrrolidone-free polycarbonate filter, with a pore size of 3 m for the neutrophil chemotaxis and 5 m for the monocyte chemotaxis, was placed over the bottom wells.…”