The RTCA method gave results very similar to the traditional method and was significantly quicker and easier to analyze.David Thirkettle-Watts and Penny Gauci (Australian Department of Defense)
Using xCELLigence for Virology Studies
Virus infection of a host cell typically includes the selective suppression of host cell functions and redirection of resources towards viral replication and assembly, ultimately leading to host cell lysis. While host cell rounding, detachment from the plate surface and/or lysis are readily detected by the xCELLigence® RTCA biosensors, more subtle changes in host cell morphology occurring during earlier phases of viral infection are also monitored.
This sensitivity to virus-induced changes in host cell morphology and behavior makes the xCELLigence® technology very well suited for a wide array of virology applications including, but not limited to: differentiating between virus strains/isolates based on the kinetics of replication and cytopathic effect, determining viral titers, determining neutralizing antibody titers, drug screening, gene therapy, and studying virus-host cell interactions using physiologically relevant cell types that cannot typically be used because they aren’t compatible with traditional assay techniques.
[The xCELLigence assay can] provide additional data when compared to classical methods. The system allowed dense real-time data collection over several days, combined with low operative effort, and avoided the danger of potentially missing significant events as may happen in end-point assays. In summary, the presented [xCELLigence-based] methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.Witkowski et al. Biochem Biophys Res Commun. 2010 Oct 8;401(1):37-41.
ACEA’s patented microtiter plates (E-Plates®) contain gold biosensors that are integrated into the bottom surface of each well. In the photo to the right, which is looking down into a single well from a 96-well plate, the array of gold biosensors is seen to cover ~75% of the well bottom’s surface area.
xCELLigence® instruments cause a miniscule electric current to flow between the E-Plate biosensors. Adherent cells act as insulators, impeding this flow of current. The ease with which this current flows is directly dependent upon the number of cells attached to the plate bottom, the size of the cells, and the cell-substrate attachment quality. Adherent cells act as insulators, impeding current flow. Importantly, side-by-side assays run in E-Plates and standard plastic microtiter plates have demonstrated that neither the gold biosensors nor the electronic monitoring have any impact on cell attachment, proliferation, etc.
Within ACEA’s E-Plates, virus-induced changes in cell morphology and attachment strength (hallmarks of a cytopathic effect) are readily detected by changes in the biosensor signal. This principle is represented schematically below, where a single well is shown at two different time points. Note that, for clarity, only two biosensors are shown in the well bottom.
Real-time monitoring of West Nile virus cytopathic effect. (A) The timing of West Nile virus (WNV)-induced cytopathic effect is dependent on virus titer. After being incubated with serial dilutions of WNV, Vero cells were seeded in the wells of an E-Plate and impedance was monitored for 200 hours. The thin horizontal line denotes CIT50 (time required for the Cell Index to decrease by 50%). (B) The time dependence of WNV-induced cytopathic effect correlates with virus titer. Plotting CIT50 as a function of known virus concentration demonstrates the strength of this correlation. Data have been adapted from J Virol Methods. 2011 May;173(2):251-8.
Key Benefits of Using xCELLigence for Virology Studies:
- Objective quantification: Subjective human observation of cytopathic effects are replaced with objective real-time data.
- Reduced workload: Once cells are infected and data acquisition has been initiated, no further involvement is required. Data is continuously recorded for anywhere from minutes to days/weeks. Plaque assays are not needed.
- Diverse applications: As long as the virus being studied has an impact on cell number, cell morphology, or cell-surface attachment strength, it can be observed by impedance-monitoring.