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Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence.

ABSTRACT: Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses, often only detecting the dominant one. Here, we present a novel flow cytometric assay that allows simultaneous assessment of cell viability and cell cycling through measurement of DNA content and DNA synthesis, and markers of cell senescence at the single cell level. We demonstrate that this assay can be performed on both human and murine cells, that are either cancerous or non-transformed, and can help to dissect complex cell fate decisions. We believe that this experimental tool will be useful for the study of diverse biological processes.

SUBMITTER: Lieschke E

PROVIDER: S-EPMC9091206 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence.

Lieschke Elizabeth E Wang Zilu Z Chang Catherine C Weeden Clare E CE Kelly Gemma L GL Strasser Andreas A

Cell death and differentiation 20220309 5

Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses ...[more]

PMID: 35264779

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Project description:Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

Dataset Information

Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence.

Publications

Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence.

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