A simple method for measuring the F-actin content of human polymorphonuclear leukocytes in whole blood

Abstract.

We have developed an improved method for measuring the filamentous (F) actin content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleton. Fresh whole blood (100 µl) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer pre-cooled to –8°C and subsequently fixed at 4°C for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugation, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothiocyanate (FITC)-phalloidin and quantified by means of flow cytometry. In healthy test subjects, PMN stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly increased F-actin assembly, while in patients with multiple organ failure, two subpopulations arose: one with greater F-actin content and a second with lower F-actin content in comparison with the unstimulated blood sample. This simple and fast method may be a useful tool in basic and clinical research.