Abstract
The Aedes albopictus Aa23 cell line, which is persistently infected with Wolbachia pipientis strain wAlbB, tends to grow as aggregated clusters of cells that are difficult to disperse for conventional quantification based on cell number. We used A. albopictus C7-10 cells to validate conversion of methylthiazole tetrazolium (MTT) to a colored formazan product with respect to incubation time, cell number over a 40-fold range, and metabolic activity as cells enter stationary phase. Using this assay, we showed that the doubling time of Aa23 cells increases from about 45 h early after plating to more than 70 h as the cells reach stationary levels. Growth of Aa23 cells proceeds at similar rates in the presence or absence of tetracycline concentrations that decrease the abundance of Wolbachia. Insofar as the MTT assay reflects mitochondrial function, our results indicate that, in Aa23 cells, abundance of intracellular Wolbachia has no measurable effect on mitochondrial activity in the presence of tetracycline.
Similar content being viewed by others
References
Ballard J. W. O.; Melvin R. G. Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila. Insect Mol. Biol 16: 799–802; 2007.
Berridge M. V.; Herst P. M.; Tan A. S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11: 127–152; 2005, doi:10.1016/S1387–2656(05)11004–7.
Cumming G.; Fidler F.; Vaux D. L. Error bars in experimental biology. J Cell Biol. 177: 7–11; 2007, doi:10.1083/jcb.200611141.
Dulbecco R.; Vogt M. Plaque formation and isolation of pure lines with poliomyelitis virus. J. Exp. Med. 99: 167–182; 1954, doi:10.1084/jem.99.2.167.
Fallon A. M. Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB. In Vitro Cell Dev. Biol. Anim. 44: 154–161; 2008.
Fallon A. M.; Kurtti T. J. Cultured cells as a tool for analysis of gene expression. In: Marquardt W. C. (ed) Biology of disease vectors. 2nd ed. Elsevier, New York, pp 539–549; 2005.
Ferree P. M.; Frydman H. M.; Li J. M.; Cao J.; Wieschaus E.; Sullivan W. Wolbachia utilizes host microtubules and dynein for anterior localization in the Drosophila oocyte. PLoS Pathogens 12: E14; 2005, doi:10.1371/journal.ppat.0010014.
Gerenday A.; Fallon A. M. Cell cycle parameters in Aedes albopictus mosquito cells. In Vitro Cell Dev. Biol. Anim. 32: 307–312; 1996, doi:10.1007/BF02723064.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55–63; 1983, doi:10.1016/0022-1759(83)90303-4.
O’Neill S. L.; Pettigrew M. M.; Sinkins S. P.; Braig H. R.; Andreadis T. G.; Tesh R. B. In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line. Insect Mol. Biol. 6: 33–39; 1997, doi:10.1046/j.1365-2583.1997.00157.x.
Schwientek M. S.; Higgins L. A.; Fallon A. M. Cultured Aedes albopictus mosquito cells accumulate elongation factor 1-α (EF-1α) during serum starvation. Insect Biochem. Mol. Biol. 32: 1055–1063; 2002, doi:10.1016/S0965-1748(02)00043-7.
Shih K. M.; Gerenday A.; Fallon A. M. Culture of mosquito cells in Eagle’s medium. In Vitro Cell Develop. Biol. Anim. 34: 629–630; 1998, doi:10.1007/s11626-996-0010-1.
Stouthamer R.; Breeuwer J. A. J.; Hurst G. D. D. Wolbachia pipientis: Microbial manipulator of arthropod reproduction. Annu. Rev. Microbiol. 53: 71–102; 1999, doi:10.1146/annurev.micro.53.1.71.
Acknowledgments
This work was supported by National Institutes of Health grant AI 070913 and by the University of Minnesota Agricultural Experiment Station, St. Paul, MN.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: J. Denry Sato
Rights and permissions
About this article
Cite this article
Fallon, A.M., Hellestad, V.J. Standardization of a colorimetric method to quantify growth and metabolic activity of Wolbachia-infected mosquito cells. In Vitro Cell.Dev.Biol.-Animal 44, 351–356 (2008). https://doi.org/10.1007/s11626-008-9129-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-008-9129-6