期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 325, 期 1-2, 页码 51-66出版社
ELSEVIER
DOI: 10.1016/j.jim.2007.05.013
关键词
cell-mediated cytotoxicity; natural killer cells (NK); lymphokine activated killer cells (LAK); flow cytometry cytotoxicity (FCC)
资金
- NIDCR NIH HHS [R01 DE013918, P01 DE012321-04, R01-DE13918, P01-DE12321, R01 DE013918-04, P01 DE012321] Funding Source: Medline
Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h Cr-51-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. (C) 2007 Elsevier B.V. All rights reserved.
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