A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry.
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 1 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.
Kasatori
Noriko
N
Department of Laboratory Medicine, Toho University School of Medicine, Ota-ku, Tokyo, Japan. kasatori@med.toho-u.ac.jp
Ishikawa
Fumio
F
Ueyama
Mamoru
M
Urayama
Tsutomu
T
D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D051017:Apoptosis Regulatory Proteins / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D048429:Cell Size; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology*; D004247:DNA / Q000737:chemistry; D004533:Egtazic Acid / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000494:pharmacology; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D011419:Propidium / Q000737:chemistry; D013194:Staining and Labeling; D053221:TNF-Related Apoptosis-Inducing Ligand; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017304:Annexin A5; D051017:Apoptosis Regulatory Proteins; D005452:Fluoresceins; D008562:Membrane Glycoproteins; D010718:Phosphatidylserines; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D011419:Propidium; D004533:Egtazic Acid; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D004247:DNA