TY - JOUR
T1 - FL-CTL assay
T2 - Fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells
AU - Chen, Kong
AU - Chen, Liansheng
AU - Zhao, Peilin
AU - Marrero, Luis
AU - Keoshkerian, Elizabeth
AU - Ramsay, Alistair
AU - Cui, Yan
PY - 2005/5/1
Y1 - 2005/5/1
N2 - 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.
AB - 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.
KW - Antigen-specific CTL function
KW - CTL assay
KW - Cytotoxic T lymphocytes
KW - Fluorescent proteins
KW - Fluorometric-based CTL
KW - GFP
UR - http://www.scopus.com/inward/record.url?scp=20444390118&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=20444390118&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2005.02.010
DO - 10.1016/j.jim.2005.02.010
M3 - Article
C2 - 15899496
AN - SCOPUS:20444390118
SN - 0022-1759
VL - 300
SP - 100
EP - 114
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -