Zamai L, Mariani A R, Zauli G, Rodella L, Rezzani R, Manzoli F A, Vitale M
Institute of Human Anatomy, University of Bologna, Italy.
Cytometry. 1998 Aug 1;32(4):280-5. doi: 10.1002/(sici)1097-0320(19980801)32:4<280::aid-cyto4>3.0.co;2-m.
Natural killer (NK) cells bind to K562 tumor target cells in vitro and kill them. The binding and cytotoxic activities of NK cells are tightly related to each other: degranulation of the cytotoxic effector is the basis for target cell damage and a consequence of effector-target recognition and binding. However, the two phases of NK activity, binding and killing, have always been measured separately by various methodologies and under different experimental conditions, because of the lack of a comprehensive methodology able to measure both of them at one time. Here we describe the simultaneous measurement of the binding and killing activities against K562 of resting and cytokine (IL-2 or IL-12)-stimulated NK cells by flow cytometry. NK, K562 and conjugates can be identified and measured by flow cytometry on the basis of NK mAb staining and target cells autofluorescence (Binding Plot). Within each population of the binding plot, killed targets can be identified and measured by their scatter characteristics (Cytotoxicity Plot). We show that i) the conjugate formation is enhanced in cytokine-stimulated cells, even at relatively short co-incubation times; ii) the conjugate release is also accelerated by cytokines; iii) the conjugate release is always quicker than the induction of the morphological changes in the target cell that generate its modified scattering properties.
自然杀伤（NK）细胞在体外可与K562肿瘤靶细胞结合并将其杀伤。NK细胞的结合活性和细胞毒性活性密切相关：细胞毒性效应物的脱颗粒是靶细胞损伤的基础，也是效应物 - 靶标识别和结合的结果。然而，由于缺乏能够同时测量NK活性两个阶段（结合和杀伤）的综合方法，这两个阶段一直是通过各种方法在不同实验条件下分别测量的。在此，我们描述了通过流式细胞术同时测量静息和细胞因子（IL-2或IL-12）刺激的NK细胞对K562的结合和杀伤活性。基于NK单克隆抗体染色和靶细胞自发荧光（结合图），可通过流式细胞术鉴定和测量NK细胞、K562细胞及其结合物。在结合图的每个群体中，可通过其散射特性（细胞毒性图）鉴定和测量被杀伤的靶细胞。我们发现：i）即使在相对较短的共孵育时间内，细胞因子刺激的细胞中结合物形成也会增强；ii）细胞因子也会加速结合物的释放；iii）结合物的释放总是比导致靶细胞形态变化并产生其改变的散射特性的诱导过程更快。
