Many viruses do not form traditional plaques, and there may not be tools readily available to determine viral foci by immunolabeling or fluorescent reporters often used for viral titration. In these cases, viral infection can be detected using induced structural changes in host cells, or cytopathic effect (CPE), including cell rounding, fusing, or other morphological changes. CPE can be detected using a label-free approach supported by the Agilent BioTek Lionheart FX Automated Microscope or Agilent BioTek Cytation Cell Imager Multimode Reader. Stitching and processing steps may be applied to achieve full-well imaging and analysis of CPE, such as decreased confluence, for each well. Using post-analysis tools in the Agilent BioTek Gen5 software, a custom binary CPE factor can be calculated for each well and used to calculate the TCID50 using the method of Reed and Muench.
Full-well images are captured in brightfield then stitched and processed as part of a fully automated Gen5 imaging protocol.
The confluency of each well is calculated and converted to a custom binary CPE factor using optimized Gen5 cellular analysis tools and post-analysis equations.
Confluency and CPE factor are reported for individual wells, and mean responses to viral dilutions are automatically graphed as part of the TCID50 calculation.
In this featured article, the authors provide a detailed description of a robust, automated method for conducting TCID50 assays to determine viral titers with the Cytation 5. This label-free imaging approach for determining cell confluence was used to identify CPE-positive and CPE-negative wells and to assess virus-based therapeutics.
The BioSpa Live Cell Analysis System enables multiplate analysis of the label-free CPE assay when integrated with the Cytation Live Cell Imager.
Agilent BioTek automated imaging systems and liquid handlers provide optimized solutions for a broad range of virology applications.
