A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.
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.
Kim
G G
GG
University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
Donnenberg
V S
VS
Donnenberg
A D
AD
Gooding
W
W
Whiteside
T L
TL
D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D019002:CD56 Antigen / Q000032:analysis; D045744:Cell Line, Tumor; D002860:Chromium Radioisotopes / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D058851:GPI-Linked Proteins; D053804:Granzymes / Q000032:analysis; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D020014:K562 Cells; D015979:Killer Cells, Lymphokine-Activated / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007694:Killer Cells, Natural / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000276:immunology; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D017452:Receptors, IgG / Q000032:analysis; D015203:Reproducibility of Results; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D000099192:CD69 Antigens
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D019002:CD56 Antigen; D002860:Chromium Radioisotopes; D003609:Dactinomycin; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D053804:Granzymes; D037181:Lectins, C-Type; D017452:Receptors, IgG; D000099192:CD69 Antigens; C428200:FCGR3B protein, human; C025942:7-aminoactinomycin D