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Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

PloS one (2012-10-25)
Ridha Limame, An Wouters, Bea Pauwels, Erik Fransen, Marc Peeters, Filip Lardon, Olivier De Wever, Patrick Pauwels
ABSTRACT

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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Gentian Violet, meets USP testing specifications
Sigma-Aldrich
Sulforhodamine B, Dye content 75 %
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Gentian Violet solution, for microscopy
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Sulforhodamine B sodium salt, powder, BioReagent, suitable for cell culture
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Sulforhodamine B sodium salt, Technical grade
Supelco
Crystal Violet, VETRANAL®, analytical standard
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Crystal Violet, indicator for the determination of the redox potential, S. No.: 785
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Crystal violet solution
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Crystal violet solution
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Crystal Violet, ACS reagent, ≥90.0% anhydrous basis
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Crystal Violet, certified by the Biological Stain Commission
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Sulforhodamine B, acid form, laser grade, Dye content 95 %
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Crystal Violet, for microscopy (Bact., Bot., Hist., Vit.), indicator (pH 0.1-2.0)
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Crystal violet solution, 1%, aqueous solution