Hashimoto K, Tatsumi N, Okuda K
Department of Laboratory Medicine, Osaka City University Medical School, Japan.
J Biochem Biophys Methods. 1989 Aug-Sep;19(2-3):143-53. doi: 10.1016/0165-022x(89)90022-5.
To examine the mechanism by which polymorphonuclear leukocytes (PMNs) move, phalloidin labelled with fluorescein isothiocyanate was introduced into freshly sampled 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 microseconds. 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 fluorescence appeared in the cytoplasm. In phagocytosis, fluorescence developed from the attachment sites, spread to the entire phagocytizing area of the cytoplasm and disappeared when phagocytosis 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.
为了研究多形核白细胞（PMNs）的运动机制，利用电细胞融合系统将异硫氰酸荧光素标记的鬼笔环肽导入新鲜采集的细胞中。最佳处理条件是施加三个100V的直流脉冲，脉冲持续时间为3微秒。在显微镜下观察时，经处理的细胞保持其正常运动能力，因此该方法适用于分析运动的活细胞。我们使用该方法研究了PMNs在运动、铺展和吞噬过程中的情况。在运动过程中，荧光首先出现在细胞头部，并沿着细胞边缘从头部逐渐向尾部移动。在铺展过程中，细胞质边缘部分周围的弥漫性荧光在细胞的附着位点和核周区域附近最强，并且在细胞质中出现荧光斑点。在吞噬过程中，荧光从附着位点开始出现，扩散到细胞质的整个吞噬区域，并在吞噬结束时消失。用细胞松弛素B处理的细胞随机出现荧光斑点。新鲜采集的细胞具有弥漫性和散在性荧光，没有在固定细胞中观察到的线条。
