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

Abstract

BackgroundCell 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 FindingsDynamic 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 IC50 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/SignificanceThe 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.

UPARANT: A Urokinase Receptor–Derived Peptide Inhibitor of VEGF-Driven Angiogenesis with Enhanced Stability and In Vitro and In Vivo Potency

“…Cell adhesion assays were performed using 16-well plates coated with 5 mg/mL vitronectin, diluted in PBS and the xCELLigence technology (Roche Diagnostics) as described (23). HUVEC (5 Â 10 3 cells/well) were plated in each coated well, in serum-free EBM in the presence or the absence of 40 ng/mL VEGF 165 with/without 10 nmol/L C-terminal amidated Ac-Glu-Arg-Phe-Arg-NH 2 control peptide (ERFR), 10 nmol/L RERF or 10 nmol/L UPAR-ANT and allowed to adhere for 4 hours at 37 C, 5% CO 2 .…”

A Urokinase Receptor–Derived Peptide Inhibiting VEGF-Dependent Directional Migration and Vascular Sprouting

“…The absorbance was determined at 570 nm using a microplate reader (Biorad). With a similar experimental design, endothelial cell proliferation was assessed using 16-well plates and the xCELLigence Technology (Roche Diagnostics) as described (31). The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a cell index value.…”

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

“…Previous studies have confirmed the validity of the real-time cell analyzer to determine cytotoxicity as well as effects on cell proliferation in cell lines in comparison with standard techniques 3,4,5,6 . For example, a good correlation was observed between readouts of the standard cell viability WST-1 assay and Cell Index values at several time points under basal proliferation conditions and after two different toxic paradigms in HeLa cells 3 .…”

“…For example, a good correlation was observed between readouts of the standard cell viability WST-1 assay and Cell Index values at several time points under basal proliferation conditions and after two different toxic paradigms in HeLa cells 3 . In A549 and MDA-MB-231 cells proliferation and cytotoxicity provoked with the microtubule stabilizer paclitaxel showed very similar values when assessed by Cell Index measurements and the standardly used sulforhodamine B (SRB) assay 4 . In the neuronal cell line of immortalized hippocampal neurons HT-22 Cell Index measurements were validated for their ability to detect cell proliferation, glutamate cytotoxicity and cytoprotection against the widely used 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium-bromide (MTT) assay 5 .…”