FL-CTL assay: fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells.
Cytotoxic T lymphocytes (CTLs) are crucial effectors against intracellular pathogens and cancer. Accurate and efficient assessment of CTL activity is important for basic and clinical studies. Widely used CTL assays, including the chromium release, JAM test and ELISPOT, involve either radioisotopes or lengthy procedures. Here, we developed a new fluorolysometric CTL assay based on cell-mediated cytolysis of fluorescent protein (GFP or DsRed) expressing cells quantified by one of the fluoro-based methods: flow cytometry, fluorescence microplate reader, or fluorescence microscopy. With flexible detection methods and lentiviral vector transduced stable lines of either GFP+ or DsRed+ cells as targets for antigen presentation and equal number of the other as internal reference for consistency and accuracy, this assay is easy to perform and to scale-up for simultaneous multi-sample analyses. Using two different antigen systems, we demonstrated that this assay is very sensitive to determine primary CTL activity of both in vitro and in vivo primed antigen-specific T cells. Thus, this FL-CTL assay is highly sensitive, reliable, reproducible, economical, convenience and supports broad applications compared to conventional CTL assays.
Chen
Kong
K
Louisiana State University Health Sciences Center, Gene Therapy Program, Department of Genetics and Medicine, 533 Bolivar Street, New Orleans, LA 70112, USA.
Chen
Liansheng
L
Zhao
Peilin
P
Marrero
Luis
L
Keoshkerian
Elizabeth
E
Ramsay
Alistair
A
Cui
Yan
Y
D000818:Animals; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods* / Q000706:statistics & numerical data; D005434:Flow Cytometry; D005470:Fluorometry / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D066298:In Vitro Techniques; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein