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Leukotoxin and its diol induce neutrophil chemotaxis through signal transduction different from that of fMLP.

When injected into animals, leukotoxin (Lx) causes acute lung injury which is associated with neutrophils infiltrating the lung tissues. However, the effect of Lx on neutrophils is still unknown, and recently it has been reported that Lx diol, a hydrolyzed metabolite, should be more potent than Lx in vitro. In this study, the authors examined the effect of Lx and its diol on human neutrophils by assessing their chemotactic response, expression of adhesion molecules, and production of peroxides. Both Lx and its diol induced chemotaxis in human neutrophils via an involvement of pertussis toxin-sensitive G-proteins, but they did not influence the expression of adhesion molecules or the production of peroxides. Furthermore, Lx synergistically affected chemotaxis with N-formyl-methionyl-leucyl-phenylalanine (fMLP), but not with endothelin-1. Neutrophil chemotaxis induced by both Lx and its diol was inhibited by phosphatidylinositol-3-kinase (PI3-K) inhibitors, but not by protein tyrosine kinase (PTK) inhibitors or by protein kinase C (PKC) inhibitors, whereas fMLP-induced chemotaxis was inhibited by PTK inhibitors, but not by PI3-K inhibitors or by PKC inhibitors. These results suggest that neutrophil chemotaxis induced by both Lx and its diol involves pathways different from those induced by fMLP. In conclusion, both leukotoxin and its diol metabolite induce chemotaxis in human neutrophils in a unique way and may act as important bioactive lipids when considering the pathological mechanism of acute lung injury.

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A Simple Technique for Reducing Edge Effect in Cell-Based Assays

Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO2 incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37°C CO2 incubator yields a significant reduction in edge effect.

While this article does not speak specifically to chemotaxis assays, it does speak generally to cell-based assays, of which chemotaxis assays are certainly part.  We are not necessarily recommending this practice, but offer it for your consideration.

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