WHOLE-BLOOD INCUBATION METHOD TO STUDY NEUTROPHIL CYTOSKELETAL DYNAMICS

To reduce artifactual effects in the study of filamentous (F)-actin dy namics in neutrophils, we have developed a whole-blood incubation meth od. Neutrophils in whole blood contained significantly less basal F-ac tin than did separated neutrophils. Although the peak relative F-actin content of neutrophils in whole blood after formyl-methionyl-leucyl-p henyl-alanine (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 fluores cence intensity and the EC,, (concentration of stimulant giving a half -maximum response). On the other hand, the EC(50) of platelet-activati ng factor (PAF) between separated neutrophils and whole-blood-incubate d neutrophils differed significantly (1.6 +/- 1.1 x 10(-9) M in separa ted neutrophils and 2.0 +/- 0.7 x 10(-8) M in whole-blood-incubated ne utrophils, p < 0.05), The whole-blood incubation method described pres ently reduces the sample volume, cost and time needed to separate neut rophils, prevents neutrophil activation during separation, and reserve s all blood components that may affect neutrophil function. For these reasons, the conditions adopted in the present method are thought to s imulate well neutrophils circulating in vivo and the method would be p referable to other neutrophil function tests performed to study actin dynamics.