Detection of baculovirus-infected insect cells by flow cytometric side-scatter analyses.
BACKGROUND
The baculovirus expression vector system (BEVS), utilizing the Autographa californica nuclear polyhedrosis virus (AcNPV), has turned out to be an attractive alternative for high-level expression (<600 mg/l) of recombinant proteins. However, there is a shortage of reliable methods for monitoring the infection process in situations where marker proteins cannot be used.


METHODS
Three recombinant baculoviruses, FastBac1-wtGFP, VTBac-GFP, and VL1392-hIL-2Ralpha, all having the genes inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), were used to infect Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-MB-0503) insect cells. The infection process of the recombinant baculoviruses was monitored by flow cytometric side-scatter and fluorescence intensity analyses over a period of 6-96 h.


RESULTS
A clear correlation between the side-scatter (SSC) signal and the relative fluorescence was observed for both of the infected cell lines, compared to noninfected cells. Comparison of SSC histograms from noninfected insect cells with cells infected with the nonfluorescent recombinant baculovirus VL1392-hIL-2Ralpha showed a clear increase of SSC for the infected cells.


CONCLUSIONS
The SSC parameter can therefore be utilized for flow cytometric monitoring of a baculovirus infection process in situations where suitable markers are not available.
Nordström
T
T
Department of Biochemistry and Pharmacy, Abo Akademi University, Abo, Finland.
Willamo
P
P
Arvela
M
M
Stenroos
K
K
Lindqvist
C
C
D000818:Animals; D016367:Baculoviridae / Q000502:physiology*; D002460:Cell Line; D002469:Cell Separation; D004279:DNA, Viral / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism / Q000821:virology; D014162:Transfection
D004279:DNA, Viral; D007202:Indicators and Reagents; D019476:Insect Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins