Whole-blood incubation method to study neutrophil cytoskeletal dynamics

Abstract

To reduce artifactual effects in the study of filamentous (F)-actin dynamics in neutrophils, we have developed a whole-blood incubation method. Neutrophils in whole blood contained significantly less basal F-actin than did separated neutrophils. Although the peak relative F-actin content of neutrophils in whole blood after formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation was significantly higher than that of separated neutrophils at 10 −9 to 10 −6 M fMLP concentrations ( p<0.05), there was no significant difference in increase in mean fluorescence intensity and the EC 50 (concentration of stimulant giving a half-maximum response). On the other hand, the EC 50 of platelet-activating factor (PAF) between separated neutrophils and whole-blood-incubated neutrophils differed significantly (1.6±1.1×10 −9 M in separated neutrophils and 2.0±0.7×10 −8 M in whole-blood-incubated neutrophils, p<0.05). The whole-blood incubation method described presently reduces the sample volume, cost and time needed to separate neutrophils, prevents neutrophil activation during separation, and reserves all blood components that may affect neutrophil function. For these reasons, the conditions adopted in the present method are thought to simulate well neutrophils circulating in vivo and the method would be preferable to other neutrophil function tests performed to study actin dynamics.