title: 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 microliters) 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 degrees C and subsequently fixed at 4 degrees 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.
authors.affiliation: Institute of Pathophysiology, Karl-Franzens University, Graz, Heinrichstr. 31, A-8010 Graz, Austria. Egger@leukotec.com
authors.forename: G
authors.initials: G
authors.lastname: Egger
authors.forename: A
authors.initials: A
authors.lastname: Burda
authors.forename: A
authors.initials: A
authors.lastname: Glasner
mesh_terms: D000199:Actins / Q000097:blood*; D000328:Adult; D003599:Cytoskeleton / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D008297:Male; D009102:Multiple Organ Failure / Q000097:blood; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000737:chemistry* / Q000187:drug effects; D010590:Phalloidine / Q000737:chemistry; D012016:Reference Values; D013194:Staining and Labeling
chemical_list: D000199:Actins; D010590:Phalloidine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016650:Fluorescein-5-isothiocyanate