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

Content Provider	Springer Nature Link
Author	Gueret, Vincent Negrete Virgen, Juan A. Lyddiatt, Andrew Al Rubeai, Mohamed
Copyright Year	2002
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.
Starting Page	87
Ending Page	97
Page Count	11
File Format	PDF
ISSN	09209069
Journal	Cytotechnology
Volume Number	38
Issue Number	1-3
e-ISSN	15730778
Language	English
Publisher	Kluwer Academic Publishers
Publisher Date	2002-01-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Biomedicine general Biochemistry Proteomics Cell Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Clinical Biochemistry Bioengineering Biomedical Engineering Biotechnology

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