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

J Immunol Methods. 2007 Aug 31;325(1-2):51-66. doi: 10.1016/j.jim.2007.05.013. Epub 2007 Jun 28.


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

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, CD / analysis
  • Antigens, Differentiation, T-Lymphocyte / analysis
  • CD3 Complex / analysis
  • CD56 Antigen / analysis
  • Cell Line, Tumor
  • Chromium Radioisotopes / metabolism
  • Cytotoxicity Tests, Immunologic / methods*
  • Dactinomycin / analogs & derivatives
  • Dactinomycin / chemistry
  • Flow Cytometry / methods*
  • Fluorescent Dyes / chemistry
  • 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
  • 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
  • Time Factors


  • Antigens, CD
  • Antigens, Differentiation, T-Lymphocyte
  • CD3 Complex
  • CD56 Antigen
  • CD69 antigen
  • Chromium Radioisotopes
  • FCGR3B protein, human
  • Fluorescent Dyes
  • GPI-Linked Proteins
  • Lectins, C-Type
  • Receptors, IgG
  • Dactinomycin
  • 7-aminoactinomycin D
  • Granzymes