Introduction of phalloidin labelled with fluorescein isothiocyanate into living polymorphonuclear leukocytes by electroporation

Abstract

To examine the mechanism by which polymorphonuclear leukocytes (PMNs) move, phalloidin labelled with fluorescein isothiocyanate was introduced into freshly sample cells by use of an electric-cell fusion system. The best conditions for treatment were three pulses of direct current at 100 V for a pulse duration of 3 μs. The treated cells retained their usual motility when observed under a microscope, so the method was suitable for the analysis of motile living cells. We used the method to study PMNs during locomotion, spreading and phagocytosis. In locomotion, fluorescence first appeared at the head of the cell and shifted gradually along the cell margin from head to tail. In spreading, diffuse fluorescence around the marginal part of the cytoplasm was strongest near both the attachment sites and the perinuclear area of the cell and spots of fluorescnece appeared in the cytoplasm. In phagocytosis, fluorescence developed from the attachment sites, spread to the entire phagocytizing area of the cytoplasma and disappeared when phagocytocis ended. Cells treated with cytochalasin B were randomly spotted with fluorescence. Freshly sampled cells had diffuse and scattered fluorescence, without the lines observed in fixed cells.