A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (<sup>51</sup>Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measure...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Claudia Coronnello, Rosalia Busà, Luca Cicero, Albert Comelli, Ester Badami
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	cytotoxic assay chromium release assay fluorescence bioluminescence imaging kinetics Photography TR1-1050 Computer applications to medicine. Medical informatics R858-859.7 Electronic computers. Computer science QA75.5-76.95
Acceso en línea:	https://doaj.org/article/983db18d08dd4486a53e322dd1f0c1b3
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:983db18d08dd4486a53e322dd1f0c1b3
record_format	dspace
spelling	oai:doaj.org-article:983db18d08dd4486a53e322dd1f0c1b32021-11-25T18:03:24ZA Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity10.3390/jimaging71102222313-433Xhttps://doaj.org/article/983db18d08dd4486a53e322dd1f0c1b32021-10-01T00:00:00Zhttps://www.mdpi.com/2313-433X/7/11/222https://doaj.org/toc/2313-433XThe cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (<sup>51</sup>Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate <sup>51</sup>Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release <sup>51</sup>Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.Claudia CoronnelloRosalia BusàLuca CiceroAlbert ComelliEster BadamiMDPI AGarticlecytotoxic assaychromium release assayfluorescencebioluminescence imagingkineticsPhotographyTR1-1050Computer applications to medicine. Medical informaticsR858-859.7Electronic computers. Computer scienceQA75.5-76.95ENJournal of Imaging, Vol 7, Iss 222, p 222 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cytotoxic assay chromium release assay fluorescence bioluminescence imaging kinetics Photography TR1-1050 Computer applications to medicine. Medical informatics R858-859.7 Electronic computers. Computer science QA75.5-76.95
spellingShingle	cytotoxic assay chromium release assay fluorescence bioluminescence imaging kinetics Photography TR1-1050 Computer applications to medicine. Medical informatics R858-859.7 Electronic computers. Computer science QA75.5-76.95 Claudia Coronnello Rosalia Busà Luca Cicero Albert Comelli Ester Badami A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
description	The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (<sup>51</sup>Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate <sup>51</sup>Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release <sup>51</sup>Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.
format	article
author	Claudia Coronnello Rosalia Busà Luca Cicero Albert Comelli Ester Badami
author_facet	Claudia Coronnello Rosalia Busà Luca Cicero Albert Comelli Ester Badami
author_sort	Claudia Coronnello
title	A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
title_short	A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
title_full	A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
title_fullStr	A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
title_full_unstemmed	A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity
title_sort	radioactive-free method for the thorough analysis of the kinetics of cell cytotoxicity
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/983db18d08dd4486a53e322dd1f0c1b3
work_keys_str_mv	AT claudiacoronnello aradioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT rosaliabusa aradioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT lucacicero aradioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT albertcomelli aradioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT esterbadami aradioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT claudiacoronnello radioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT rosaliabusa radioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT lucacicero radioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT albertcomelli radioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity AT esterbadami radioactivefreemethodforthethoroughanalysisofthekineticsofcellcytotoxicity
_version_	1718411696164306944

A Radioactive-Free Method for the Thorough Analysis of the Kinetics of Cell Cytotoxicity

Ejemplares similares