Development and characterization of a novel luciferase based cytotoxicity assay

Matta, Hittu; Gopalakrishnan, Ramakrishnan; Choi, Sunju; Prakash, Rekha; Natarajan, Venkatesh; Prins, Ruben; Gong, Songjie; Chitnis, Saurabh Deepak; Kahn, Michael; Han, Xu; Chaudhary, Vishan; Soni, Adam; Sernas, Jennifer; Khan, Prottasha; Wang, Dan; Chaudhary, Preet M.

doi:10.1038/s41598-017-18606-1

Cited by 45 publications

(59 citation statements)

References 29 publications

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“…Various assays have been developed that assess the release of cell components from damaged or dead cells such as radioactive chromium, enzymes, and cytokines after exposure to activated T cells 13,[16][17][18][19] or that measure targets labeled with fluorescent dyes. [20][21][22] Indirect methods such as measurement of interferon gamma or granzyme B release by T cells or intracellular granzyme and perforin upregulation in T cells assess the activation of effector T cells but not target cell killing.…”

Section: Discussionmentioning

confidence: 99%

“…14,15 Multiple assays that quantify the release of cell components from damaged target cells or use flow cytometric analysis have also been developed to assess cell-mediated cytotoxicity. [16][17][18][19][20][21][22] Unfortunately, these assays have caveats such as requiring high cell viability or not providing data for multiple timepoints. In addition, many of these methods rely on cells expressing reporter genes and would be difficult to adapt for primary T cells.…”

Section: Introductionmentioning

confidence: 99%

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Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell

Haeseleer

Eichholz

Tareen³

et al. 2020

AIDS Research and Human Retroviruses

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The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell

Haeseleer

Eichholz

Tareen³

et al. 2020

AIDS Research and Human Retroviruses

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“…The Firefly luciferase (Fluc) expression plasmid pLenti-EF1a-Pac-T2A-Fluc was generated by replacing the Gaussia luciferase (Gluc) of the pLenti-EF1a-Pac-T2A-Gluc plasmid [15] (kindly provided by Preet Chaudhary, University of Southern California, CA, USA) with the Firefly Luciferase hluc + (Promega, Waltham, MA, USA) via Gibson Assembly (NEB).…”

Section: Plos Onementioning

confidence: 99%

A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences

et al. 2020

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To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of the human αβTCR constant regions with their codon-optimized murine counterparts for chimerization, addition of a second interchain disulfide bond and arrangement of the TCR chains in the order β-P2A-α. We exemplified the utility of our vector backbone by cloning and functional testing of three melanoma-reactive TCRs in primary human T cells.

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“…Since CopLucs show a high thermostability, the MLuc was applied as a reporter in studies on cellular hyperthermia at nanoparticle‐mediated heating . With CopLucs as a basis, the biosensors to detect cellular endoplasmic reticulum stress and caspase activation , to evaluate the metabolic activity and cell viability in real time and to assess cytotoxicity were constructed .…”

Section: Application Of Copepod Luciferasesmentioning

confidence: 99%

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

Photochem & Photobiology

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Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

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Development and characterization of a novel luciferase based cytotoxicity assay

Cited by 45 publications

References 29 publications

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell

A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

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