Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays
| Autor: | Wei-Chiang Chen, Carl Co, Xiaohui Lu, Emily Menesale, Svetlana Bergelson, Allyson Masci |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Virus quantification Chemistry fluorescent detection plaque assay Limiting gene therapy Stain Fluorescence Article image-based counting 03 medical and health sciences automated counting 030104 developmental biology 0302 clinical medicine Robustness (computer science) 030220 oncology & carcinogenesis Genetics assay robustness Molecular Medicine Sensitivity (control systems) Molecular Biology Image based Biomedical engineering |
| Zdroj: | Molecular Therapy. Methods & Clinical Development |
| ISSN: | 2329-0501 |
| DOI: | 10.1016/j.omtm.2019.07.007 |
| Popis: | Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation. |
| Databáze: | OpenAIRE |
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