Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement

Based on the enhancement of rhodamine phalloidin fluorescence after its bin ding to actin filaments we have developed a technique to quantify F-actin, drastically (much greater than 100 times) reducing consumption of the expen sive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation tim es and stabilization of the filaments by actin-binding proteins or formalde hyde. Equilibrium and kinetic mathematical models relating rhodamine fluore scence with F-actin concentrations were used to predict the optimal assay c onditions. The method has been applied to measure relative and absolute F-a ctin concentrations in cytosolic fractions and stimulus-induced actin polym erization in neutrophils. The cells were lysed with octyl-beta-D-glucopyran oside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previous ly required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Mor eover, the method is unique for reliable and easy F-actin measurements in c ell-free systems. (C) 1998 Academic Press.