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

We have developed an improved method for measuring the filamentous (F) acti n content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleto n. Fresh whole blood (100 mul) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer preco oled to -8 degreesC and subsequently fixed at 4 degreesC for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugati on, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothi ocyanate (FITC)phalloidin and quantified by means of flow cytometry. In hea lthy test subjects, PMN stimulation by the chemotactic peptide formyl-methi onyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly incre ased F-actin assembly, while in patients with multiple organ failure, two s ubpopulations arose: one with greater F-actin content and a second with low er F-actin content in comparison with the unstimulated blood sample. This s imple and fast method may be a useful tool in basic and clinical research.