Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy

✍ Scribed by Mary L. Musco; Shijun Cui; David Small; Margarita Nodelman; Barry Sugarman; Michael Grace

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 127 KB
• Volume: 33
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19981101)33:3<290::aid-cyto2>3.0.co;2-l

✦ Synopsis

The determination of recombinant adenoviral (rAd) infectivity and p53 protein expression is important for the evaluation of rAd vectors containing the p53 gene (rAd-CMV-p53) for gene therapy. We have previously reported that rAd5-CMV-p53 vectors can be assessed for infectivity and concomitant p53 protein expression in single-and two-color assays using intracellular staining methodology and flow cytometric analysis. We have compared the flow cytometry-based assays for rAd infectivity (hexon protein) and p53 protein expression with the new slide-based laser scanning cytometry (LSC). We report that LSC analysis of rAd-CMV-p53-infected human 293 cells correlated very well with flow cytometric analysis across a wide range of viral infectivity for both infectivity assessment (r 2 ‫؍‬ 0.97) and p53 protein expression (r 2 ‫؍‬ 0.96). Absolute values for infective titer and p53 protein expression titer from an rAd5-CMV-p53 production batch were similar and within experimental error with the two different analytical methods. Finally, bivariate format analysis of rAd-CMV-p53-infected cells revealed comparable results between LSC and flow cytometric analysis. LSC is a reliable and useful tool for the intracellular staining for adenoviral hexon protein expression for determining infectivity and for p53 protein expression from an expressed p53 transgene.