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      Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

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          Abstract

          Background

          Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions.

          Methodology/Principal Findings

          Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC 50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method.

          Conclusions/Significance

          The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.

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          Most cited references10

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          THE CHEMOTACTIC EFFECT OF MIXTURES OF ANTIBODY AND ANTIGEN ON POLYMORPHONUCLEAR LEUCOCYTES

          An in vitro technique is described for assessing the chemotactic activity of soluble substances on motile cells. Antibody-antigen mixtures when incubated (37°C) in medium containing fresh (i.e. non-inactivated) normal rabbit serum exert a strong chemotactic effect on rabbit polymorphonuclear leucocytes. Results are described which indicate that, when antibody-antigen complexes are incubated (37°C) in fresh serum, a heat-stable (56°C) substance (or substances) is produced which acts directly as a chemotactic stimulus on the polymorphs. This heat-stable chemotactic substance is not produced when antibody-antigen complexes are incubated in serum which has been heated at 56°C for 30 minutes.
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            The xCELLigence system for real-time and label-free monitoring of cell viability.

            We describe here the use of the xCELLigence system for label-free and real-time monitoring of cell -viability. The xCELLigence system uses specially designed microtiter plates containing interdigitated gold microelectrodes to noninvasively monitor the viability of cultured cells using electrical impedance as the readout. The continuous monitoring of cell viability by the xCELLigence system makes it possible to distinguish between different perturbations of cell viability, such as senescence, cell toxicity (cell death), and reduced proliferation (cell cycle arrest). In addition, the time resolution of the xCELLigence system allows for the determination of optimal time points to perform standard cell viability assays as well as other end-point assays to understand the mode of action. We have used the WST-1 assay (end-point viability readout), the cell index determination (continuous monitoring of viability by xCELLigence), and the DNA fragmentation assay (end-point apoptosis assay) to systematically examine cytotoxic effects triggered by two cytotoxic compounds with different cell-killing kinetics. Good correlation was observed for viability readouts between WST-1 and cell index. The significance of time resolution by xCELLigence readout is exemplified by its ability to pinpoint the optimal time points for conducting end point viability and apoptosis assays.
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              Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects.

              We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This noninvasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds, and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRPs). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor celecoxib, and identified an additional mechanism for the experimental compound monastrol. We also identified and characterized a new antimitotic agent. Our findings indicate that the TCRP approach provides predictive mechanistic information for small molecule compounds.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                19 October 2012
                : 7
                : 10
                : e46536
                Affiliations
                [1 ]Center for Oncological Research (CORE), University of Antwerp, Antwerp, Belgium
                [2 ]StatUA Center for Statistics, University of Antwerp, Antwerp, Belgium
                [3 ]Department of Oncology, Antwerp University Hospital, Edegem (Antwerp), Belgium
                [4 ]Laboratory of Experimental Cancer Research, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, Ghent, Belgium
                [5 ]Laboratory of Pathology, Antwerp University Hospital, Edegem (Antwerp), Belgium
                Wayne State University School of Medicine, United States of America
                Author notes

                Competing Interests: Olivier De Wever, listed as co-author, is currently serving as an academic editor for PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: RL AW BP EF MP FL ODW PP. Performed the experiments: RL AW BP. Analyzed the data: RL AW BP EF. Contributed reagents/materials/analysis tools: MP FL PP. Wrote the paper: RL. Evaluated and interpreted results: RL AW BP EF MP FL ODW PP. Evaluated manuscript text: AW EF MP FL ODW PP.

                Article
                PONE-D-12-11765
                10.1371/journal.pone.0046536
                3477108
                23094027
                3dbfb4c6-b27a-4d0f-bfb4-71dea1622a16
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 24 April 2012
                : 31 August 2012
                Page count
                Pages: 12
                Funding
                These authors have no support or funding to report.
                Categories
                Research Article
                Biology
                Biophysics
                Cell Motility
                Developmental Biology
                Morphogenesis
                Cell Migration
                Cell Differentiation
                Molecular Cell Biology
                Cell Growth
                Medicine
                Diagnostic Medicine
                Pathology
                Anatomical Pathology
                Cytopathology
                Test Evaluation
                Oncology
                Basic Cancer Research
                Cancers and Neoplasms

                Uncategorized
                Uncategorized

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