Cargando…

A real-time, bioluminescent annexin V assay for the assessment of apoptosis

Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pat...

Descripción completa

Detalles Bibliográficos
Autores principales: Kupcho, Kevin, Shultz, John, Hurst, Robin, Hartnett, Jim, Zhou, Wenhui, Machleidt, Thomas, Grailer, Jamison, Worzella, Tracy, Riss, Terry, Lazar, Dan, Cali, James J., Niles, Andrew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373262/
https://www.ncbi.nlm.nih.gov/pubmed/30498998
http://dx.doi.org/10.1007/s10495-018-1502-7
_version_ 1783394948590075904
author Kupcho, Kevin
Shultz, John
Hurst, Robin
Hartnett, Jim
Zhou, Wenhui
Machleidt, Thomas
Grailer, Jamison
Worzella, Tracy
Riss, Terry
Lazar, Dan
Cali, James J.
Niles, Andrew
author_facet Kupcho, Kevin
Shultz, John
Hurst, Robin
Hartnett, Jim
Zhou, Wenhui
Machleidt, Thomas
Grailer, Jamison
Worzella, Tracy
Riss, Terry
Lazar, Dan
Cali, James J.
Niles, Andrew
author_sort Kupcho, Kevin
collection PubMed
description Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death.
format Online
Article
Text
id pubmed-6373262
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Springer US
record_format MEDLINE/PubMed
spelling pubmed-63732622019-03-01 A real-time, bioluminescent annexin V assay for the assessment of apoptosis Kupcho, Kevin Shultz, John Hurst, Robin Hartnett, Jim Zhou, Wenhui Machleidt, Thomas Grailer, Jamison Worzella, Tracy Riss, Terry Lazar, Dan Cali, James J. Niles, Andrew Apoptosis Article Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death. Springer US 2018-11-29 2019 /pmc/articles/PMC6373262/ /pubmed/30498998 http://dx.doi.org/10.1007/s10495-018-1502-7 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Article
Kupcho, Kevin
Shultz, John
Hurst, Robin
Hartnett, Jim
Zhou, Wenhui
Machleidt, Thomas
Grailer, Jamison
Worzella, Tracy
Riss, Terry
Lazar, Dan
Cali, James J.
Niles, Andrew
A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title_full A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title_fullStr A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title_full_unstemmed A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title_short A real-time, bioluminescent annexin V assay for the assessment of apoptosis
title_sort real-time, bioluminescent annexin v assay for the assessment of apoptosis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373262/
https://www.ncbi.nlm.nih.gov/pubmed/30498998
http://dx.doi.org/10.1007/s10495-018-1502-7
work_keys_str_mv AT kupchokevin arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT shultzjohn arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT hurstrobin arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT hartnettjim arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT zhouwenhui arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT machleidtthomas arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT grailerjamison arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT worzellatracy arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT rissterry arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT lazardan arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT calijamesj arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT nilesandrew arealtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT kupchokevin realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT shultzjohn realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT hurstrobin realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT hartnettjim realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT zhouwenhui realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT machleidtthomas realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT grailerjamison realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT worzellatracy realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT rissterry realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT lazardan realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT calijamesj realtimebioluminescentannexinvassayfortheassessmentofapoptosis
AT nilesandrew realtimebioluminescentannexinvassayfortheassessmentofapoptosis