Cell-based actin polymerization assay to analyze chemokine inhibitors

Abstract

Chemokines play an important role in various diseases as signaling molecules for immune cells. Therefore, the inhibition of the chemokine-receptor interaction and the characterization of potential inhibitors are important steps in the development of new therapies. Here, we present a new cell-based assay for chemokine-receptor interaction, using chemokine-dependent actin polymerization as a readout. We used interleukin-8 (IL-8, CXCL8) as a model chemokine and measured the IL-8-dependent actin polymerization with Atto565-phalloidin by monitoring the fluorescence intensity in the cell layer after activation with IL-8. This assay needs no transfection, is easy to perform and requires only a few working steps. It can be used to confirm receptor activation and to characterize the effect of chemokine receptor antagonists. Experiments with the well-known CXCR1/2 inhibitor reparixin confirmed that the observed increase in fluorescence intensity is a result of chemokine receptor activation and can be inhibited in a dose-dependent manner. With optimized parameters, the difference between positive and negative control was highly significant and statistical Z´-factors of 0.4 were determined on average.

Keywords: Actin polymerization; Cell-based assay; Chemokine inhibitors; Chemokines; Reparixin.