Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 307, Issue 1-2, Pages 41-53Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2005.09.005
Keywords
natural killer; 2 ',7 '-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF); Annexin V; propidium iodide; cytotoxicity; apoptosis
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Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2',7'-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage I to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity. (c) 2005 Elsevier B.V. All rights reserved.
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