The Agilent xCELLigence RTCA eSight workflow provides a simple way to measure, quantify, and monitor the kinetics of 3D tumor spheroid growth or shrinkage by live cell imaging using a combination of brightfield plus red, green, and blue fluorescence. The RTCA eSight suite of software tools enables drug efficacy (IC50) to be calculated based on diverse spheroid metrics such as area, diameter, and brightness. Briefly, cells are first seeded into a round bottom ULA plate, and cell nuclei can be labeled using either the Agilent lentivirus reagents (eLenti) or live cell dyes (eLive). Multiplexing is also possible using Agilent eTox dyes to detect dead cells, or Agilent eCaspase and eAnnexin reagents to specifically monitor apoptosis. The eSight is currently the only instrument that provides both impedance and live cell imaging readouts of cell health and behavior in parallel, providing multiple perspectives in one workflow that may otherwise be missed.
 

Gain deeper and more physiologically relevant information - as 3D better mimics and models the tumor microenviroment (with respect to drug resistance), while having many distinct advantages over traditional 2D cellular assays. 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. 3D spheroids exhibit numerous physiological traits such as increased cell survival, a hypoxic core, and a more relevant morphology. With tumor spheroid assays on the RTCA eSight- monitor and quantify growth (either label or label-free) of 3D single tumor spheroids – all inside your incubator. Reveal cellular changes (i.e. morphology) over time in either mono or co-culture models, and uncover drug mechanism(s) of action. The RTCA eSight provides a real-time solution for simple 3D cellular viability or toxicity readouts, generating reproducible data, and deriving deeper and more meaningful biological insights into your drug discovery assays.

As shown here (video), SKOV3 human ovarian adenocarcinoma cells (red nuclei labeled) are treated with and without Taxol 3.7 nM, and monitored for up to 10 days. Upon taxol treatment, cell death is then monitored and measured by eCaspase Green. Cell death along with tumor growth or shrinkage are easily quantified and measured in real-time. Bright-field channel data analysis parameters include Diameter (µm) and Total Area (µm2/Image); Fluorescence channel data analysis parameters include: Diameter (µm), Total Area (µm2/Image), Mean Intensity (RI) and Total Integrated Intensity (RI*µm2). Red area is representative of spheroid shape; Red intensity can be representative of cell viability; whereas Green intensity is a measurement of cytotoxicity. For more information regarding quantification and data output, please see the latest 3D Spheroid App Note for more information featured on: Agilent.com/chem/esight