FL-CTL assay: Fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells

Chen, Kong; Chen, Liansheng; Zhao, Peng; Marrero, Luis; Keoshkerian, Elizabeth; Ramsay, Alistair J.; Cui, Yan

doi:10.1016/j.jim.2005.02.010

Cited by 22 publications

(18 citation statements)

References 24 publications

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“…Effects of primed DCs and CTLs on target cells have been clearly shown in previous studies 8,12,15. To determine changes in target cells, particularly cytotoxicity, distinct methods based on the properties of target cells undergoing apoptosis have been used to measure products or amounts of target cells killed by CTLs 8,12,15.…”

Section: Discussionmentioning

confidence: 91%

“…To determine changes in target cells, particularly cytotoxicity, distinct methods based on the properties of target cells undergoing apoptosis have been used to measure products or amounts of target cells killed by CTLs 8,12,15. When target cells die, they release intracellular proteins such as granzyme B, CD107a, and caspase-3.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it is important to develop nonradioactive methods to replace the 51Cr assay. To evaluate the effects of CTLs on target cells, previous studies8–12 labeled the target cells or effector cells with fluorescent dyes. The cytotoxicity value was recorded as the dead cell population appearing after incubation with CTLs 10.…”

Section: Introductionmentioning

confidence: 99%

“…The cytotoxicity value was recorded as the dead cell population appearing after incubation with CTLs 10. Other studies transduced target cells with a gene encoding green fluorescent protein11,12 or a combination of GFP and luciferase 8. Another method is based on the expression of caspase 3 in target cells after contact with CTLs, wherein CTL-mediated cytotoxicity is determined by flow cytometry to evaluate the caspase 3 positive population 13,14.…”

Section: Introductionmentioning

confidence: 99%

“…From these studies, the cytotoxicity of CTLs was calculated 15. Although most of these assays have some advantages, such as simplicity, and have a sensitivity similar or higher to 51Cr,10,12–15 they also have some limitations, especially when the effect of CTLs on target cells is non-continuously recorded and monitored in real time. Furthermore, the methods based on flow cytometry require sufficient numbers of target cells for evaluation.…”

Section: Introductionmentioning

confidence: 99%

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A simple in vitro method for evaluating dendritic cell-based vaccinations

Pham¹,

Nguyen²,

Nguyen³

et al. 2014

OTT

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BackgroundDendritic cell (DC) therapy is a promising therapy for cancer-targeting treatments. Recently, DCs have been used for treatment of some cancers. We aimed to develop an in vitro assay to evaluate DC therapy in cancer treatment using a breast cancer model.MethodsDCs were induced from murine bone marrow mononuclear cells in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Immature DCs were primed with breast cancer stem cell (BCSC)-derived antigens. BCSCs were sorted from 4T1 cell lines based on aldehyde dehydrogenase expression. A mixture of DCs and cytotoxic T lymphocytes (CTLs) were used to evaluate the inhibitory effect of antigen-primed DCs on BCSCs. BCSC proliferation and doubling time were recorded based on impedance-based cell analysis using the xCELLigence system. The specification of inhibitory effects of DCs and CTLs was also evaluated using the same system.ResultsThe results showed that impedance-based analysis of BCSCs reflected cytotoxicity and inhibitory effects of DCs and CTLs at 72 hours. Differences in ratios of DC:CTL changed the cytotoxicity of DCs and CTLs.ConclusionThis study successfully used impedance-based cell analysis as a new in vitro assay to evaluate DC efficacy in cancer immunotherapy. We hope this technique will contribute to the development and improvement of immunotherapies in the near future.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A simple in vitro method for evaluating dendritic cell-based vaccinations

Pham¹,

Nguyen²,

Nguyen³

et al. 2014

OTT

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Methods and Applications of Cytogenetic Receptor and Enzyme Assays

Peng

2009

Pharmaceutical Bioassays

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Cytotoxicity of CD19-CAR-NK92 cells is primarily mediated via perforin/granzyme pathway

Althaus

Nilius‐Eliliwi

Maghnouj

et al. 2023

Cancer Immunol Immunother

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Chimeric antigen receptors (CARs) have improved cancer immunotherapy in recent years. Immune cells, such as Natural killer cells (NK-cells) or T cells, are used as effector cells in CAR-therapy. NK92-cells, a cell line with known cytotoxic activity, are of particular interest in CAR-therapy since culturing conditions are simple and anti-tumor efficacy combined with a manageable safety profile was proven in clinical trials. The major pathways of immune effector cells, including NK92-cells, to mediate cytotoxicity, are the perforin/granzyme and the death-receptor pathway. Detailed knowledge of CAR-effector cells’ cytotoxic mechanisms is essential to unravel resistance mechanisms, which potentially arise by resistance against apoptosis-inducing signaling. Since mutations in apoptosis pathways are frequent in lymphoma, the impact on CAR-mediated cytotoxicity is of clinical interest. In this study, knockout models of CD19-CAR-NK92 cells were designed, to investigate cytotoxic pathways in vitro. Knockout of perforin 1 (Prf1) and subsequent abrogation of the perforin/granzyme pathway dramatically reduced the cytotoxicity of CD19-CAR-NK92 cells. In contrast, knockout of FasL and inhibition of TRAIL (tumor necrosis factor-related apoptosis-inducing ligands) did not impair cytotoxicity in most conditions. In conclusion, these results indicate the perforin/granzyme pathway as the major pathway to mediate cytotoxicity in CD19-CAR-NK92 cells.

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FL-CTL assay: Fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells

Cited by 22 publications

References 24 publications

A simple in vitro method for evaluating dendritic cell-based vaccinations

A simple in vitro method for evaluating dendritic cell-based vaccinations

Methods and Applications of Cytogenetic Receptor and Enzyme Assays

Cytotoxicity of CD19-CAR-NK92 cells is primarily mediated via perforin/granzyme pathway

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