A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

• Published in: Journal of immunological methods Vol. 325; no. 1; pp. 51 - 66
• Main Authors: Kim, G.G., Donnenberg, V.S., Donnenberg, A.D., Gooding, W., Whiteside, T.L.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 31.08.2007; Elsevier
• Subjects: Antigens, CD - analysis; Antigens, Differentiation, T-Lymphocyte - analysis; Biological and medical sciences; CD3 Complex - analysis; CD56 Antigen - analysis; Cell Line, Tumor; Cell-mediated cytotoxicity; Chromium Radioisotopes - metabolism; Cytotoxicity Tests, Immunologic - methods; Dactinomycin - analogs & derivatives; Dactinomycin - chemistry; Flow Cytometry - methods; Flow cytometry cytotoxicity (FCC); Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; GPI-Linked Proteins; Granzymes - analysis; Humans; Immunophenotyping - methods; K562 Cells; Killer Cells, Lymphokine-Activated - chemistry; Killer Cells, Lymphokine-Activated - immunology; Killer Cells, Lymphokine-Activated - metabolism; Killer Cells, Natural - chemistry; Killer Cells, Natural - immunology; Killer Cells, Natural - metabolism; Kinetics; Lectins, C-Type; Leukocytes, Mononuclear - immunology; Lymphokine activated killer cells (LAK); Molecular immunology; Natural killer cells (NK); Neoplasms - immunology; Neoplasms - metabolism; Neoplasms - pathology; Receptors, IgG - analysis; Reproducibility of Results; T-Lymphocyte Subsets - chemistry; T-Lymphocyte Subsets - immunology; T-Lymphocyte Subsets - metabolism; T-Lymphocytes, Cytotoxic - chemistry; T-Lymphocytes, Cytotoxic - immunology; T-Lymphocytes, Cytotoxic - metabolism; Techniques; Time Factors; Lymphokine activated killer cells (LAK); Cell-mediated cytotoxicity; Flow cytometry cytotoxicity (FCC); Natural killer cells (NK); Natural killer cell; Flow cytometry; Immunophenotype; LAK cell; Cytotoxicity; Release; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2007.05.013

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4–16 h 51Cr-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.