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. 2002 Jan;38(1-3):87–97. doi: 10.1023/A:1021106116887

Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells

Vincent Gueret 1, Juan A Negrete-Virgen 1, Andrew Lyddiatt 1, Mohamed Al-Rubeai 1,✉
PMCID: PMC3449930  PMID: 19003090

Abstract

There is a constant and growing interest in exploitingadenoviruses as vectors for gene therapy when transientexpression of a therapeutic protein is necessary. Therequirement for an increased viral titre has prompted asearch for techniques by which this virus may be assayedwith greater speed and simplicity. Conventional plaqueassay for quantification of adenoviral vectors titre incurrent use is laborious and time-consuming (up to 14days). We report herein a method for the monitoring ofadenovirus expressing green fluorescent protein thatincorporates rapid and easy sample handling by means offlow cytometric analysis. Cells (HEK293) were infectedwith adenovirus at various multiplicity of infection(MOI), harvested 17 to 20 h post infection and analysedby flow cytometry. Assumptions were made that onefluorescent cell was infected by a single infectiousparticle at a relatively low MOI. The adenoviral titrewas subsequently estimated from cell analysis in arelatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with thatobtained using a non-complementing cell line (A549). APoisson distribution successfully modelled the profile ofinfection as a function of MOI. This provided a betterunderstanding of adenoviral infection at the earlieststage possible. Monitoring of GFP fluorescence and viruspropagation in a batch culture of infected cells wassubsequently used as a practical application of thevalidated method.

Keywords: adenovirus titration, adenoviral vectors, flowcytometry, GFP, infectivity

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