Baculoviruses have become powerful tools for a growing number of applications, such as the overproduction of recombinant proteins in insect larvae, insect cells, and mammalian cells, the surface display of peptides or proteins, and the potential for use as safe vectors for gene therapy (1,2). The precise titration of virus stocks is a prerequisite for the optimization of protein expression in insect and mammalian cells (3). The accuracy of the resulting titer is determined by the precision and the reproducibility of the method employed. Unfortunately, commonly applied methods (plaque assay and end-point dilution assay) are known to produce variable results, are laborious to perform, and require long incubation times (4). Recently, the variability of virus titer determinations has been shown to be reduced by coexpression of reporter proteins, such as β-galactosidase (5) or green fluorescent protein (6,7,8) to differentiate infected from noninfected cells. However, the coexpression of a reporter protein is not always desirable for target protein expression because it may reduce the expression levels of the protein of interest. Other methods have been developed that use antibodies against baculoviral proteins (9), and commercial immunostaining kits are available [e.g., the FastPlax™ Titer Kit (EMD Biosciences, Madison, WI, USA) and the BD BacPAK™ Baculovirus Rapid Titer Kit (Clontech, Mountain View, CA, USA)]. However, these methods necessitate laborious data collection processes and, in our hands, are subject to high user-to-user variation for baculovirus titer determination.

The fastest methods reported are based on viral DNA quantitation using either flow cytometry (10) or real-time PCR (11), which allow titer determination within two hours. The drawbacks of these approaches are the costs of equipment and staining reagents, and the fact that the total number of particles and not the number of infectious particles is determined.

An alternative approach is based on the lytic nature of the viral system (12), and more specifically on the fact that cell growth is attenuated upon virus infection. This growth reduction is dose-dependent and can be estimated by measuring the viable cell concentration and subsequently correlating this to the virus titer. Indeed, a new method was recently developed for virus titration by spectrophotometrically monitoring the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (4). The accuracy of the method was clearly demonstrated; however, the number of preparation steps and the overall duration (6 days) are not compatible with a fast and automated high-throughput process.

In this work, we demonstrate that AlamarBlue™ (Serotec, NC, USA) is a powerful substitute for MTT to monitor the early cell growth arrest induced by a 24-h baculovirus infection cycle. AlamarBlue is a sensitive oxidation-reduction indicator; the fluorescent color change upon reduction by living cells is believed to be mediated by mitochondrial enzymes (13). AlamarBlue has been shown to be useful for measuring the proliferation of several eukaryotic cell lines (14,15,16,17). However, to our knowledge, no attempt was made to use AlamarBlue to assess insect cell proliferation.

In the first step, we investigated the correlation between viable insect cell number and fluorescence intensity. A suspension culture of Spodoptera frugiperda Sf21 cells (Invitrogen, Carlsbad, CA, USA) in mid-exponential growth phase was used to set up the assay. The cells were grown in ExCell 401™ cultivation medium (JRH Biosciences, Lenexa, KS, USA) supplemented with 1% fetal calf serum (FCS) and 2 mM glutamine, ensuring a cell doubling time of 22 ± 2 h and viability higher than 94%. From 3.75 × 10² to 6.0 × 10³ viable cells per well were seeded in a flat-bottomed 96-well plate (BD Falcon™; BD Biosciences, San Jose, CA, USA) in 200 µL culture volume. To avoid the variations inherent to the plate side effects, wells at the edges of the plate were not used for measurements. After the addition of 10% AlamarBlue (v/v), the plate was incubated at 28°C, and fluorescence was measured after 3–24 h using a fluorescence plate reader (CytoFluor® II; Applied Biosystems, Foster City, CA, USA). The excitation wavelength was set to 530 nm and the emission wavelength to 590 nm. This correlation proved to be linear for cell concentrations up to 4.5 × 10³ cells per well with a correlation coefficient of R = 0.9995. A slight decrease of linearity is observed at cell concentrations above 4.5 × 10³ cells per well (R = 0.9743), indicating that a saturation response is reached above the tested cell concentrations. We therefore chose to seed 3 × 10³ cells per well and a measurement of fluorescence 5 h after the addition of AlamarBlue because these conditions ensure a reliable linear response even if the cells are doubling during the incubation time.