FLARE: A Flow Cytometry–Based Fluorescent Assay for Measuring HSV-1 Nuclear Egress

During herpesvirus replication, capsids are assembled inside the nucleus and translocated into the cytosol by a non-canonical nucleocytoplasmic export process termed <i>nuclear egress</i>. Traditional methods of measuring nuclear egress rely on imaging-based technologies such as confocal and electron microscopy. These techniques are labor-intensive, limited by the number of cells that can be examined, and may not accurately represent the entire population, generating a potential bias during data interpretation. To overcome these problems, we have developed a flow cytometry-based method to measure HSV-1 nuclear egress that we termed FLARE (FLow cytometry-based Assay of nucleaR Egress). This assay uses a double fluorescent reporter system, utilizing HSV-1-tdTomato to identify infected cells and an Alexa Fluor-488-conjugated, capsid-specific antibody to detect cytosolic capsids, thereby distinguishing infected cells with nuclear egress from those without it. This assay provides more quantitative results than traditional methods, enables large-scale high throughput, and can be adapted for use with other herpesviruses. Key features • Quantification of HSV-1 nuclear egress by flow cytometry using a double fluorescent reporter system. • The assay is suitable for large-scale high-throughput screens, e.g., CRISPR/Cas9. • The assay can be adapted for use with other herpesviruses, provided a mature capsid-specific antibody is available.