Summary
A new cytotoxicity assay has been developed to assess the activity of the toxins produced byBacillus thuringiensis kurstaki on CFl cells. The assay is performed in a 96-well plate using a tetrazolium salt, MTT, as the substrate. The compound diffuses through the cell membrane and is reduced by mitochondrial dehydrogenases of live cells to a purple color compound, which precipitates within the cells. The precipitate is dissolved by isopropanol and the absorbance at 570 nm is measured with a 96-well plate reader. The amount of dye converted is proportional to the number of liver cells and the length of incubation with substrate. The LC50 of the 60 kDa toxin, an activated form of the 130 kDaB. t. kurstaki toxin has been determined and found to be comparable to the data obtained by the trypan blue staining of dead cells.
Similar content being viewed by others
References
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Bioch. 72:248–254; 1976.
Bulla, L. A., Jr.; Davidson, L. I.; Kramer, K. J., et al. Purification of the insecticidal toxin from the parasporal crystal ofBacillus thuringiensis subsp.kurstaki. Bioch. Biophy. Res. Comm. 91:1123–1130; 1979.
English, L. H.; Cantley, L. C. Delta endotoxin inhibits Rb+ uptake, lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells. J. Memb. Biol. 85:199–204; 1985.
Fast, P. G.; Murphy, D. W.; Sohi, S. S.Bacillus thuringiensis d-endotoxin: evidence that toxin acts at the surface of susceptible cells. Experientia 34:762–763; 1976.
Gill, S. S.; Hornung, J. M. Cytolytic activity ofBacillus thuringiensis proteins to insect and mammalian cell lines. J. Invert. Path. 50:16–25; 1987.
Green, L. M.; Reade, J. L.; Ware, C. F. Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70:257–268; 1984.
Haider, M. Z.; Knowles, B. H.; Ellar, D. J. Specificity ofBacillus thuringiensis var.colmeri insecticidal δ-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. Eur. J. Biochem. 156:531–540; 1986.
Johnson, D.; Davidson, L. I. Specificity of cultured insect tissue cells for bioassay of entomocidal protein fromBacillus thuringiensis. In Vitro 20:66–70; 1984.
Johnson, D. E. Incidence of insect cell cytolytic activity amongBacillus thuringiensis serotypes. FEMS Microbiol. Lett. 43:121–125; 1987.
Knowles, B. H.; Ellar, D. J. Colloid-osmotic lysis is a general feature of the mechanism of action ofBacillus thuringiensis δ-endotoxins with different insect specificity. Biochem. Biophys. Acta 924:509–518; 1987.
Knowles, B. H.; Thomas, W. E.; Ellar, D. J. Lectin-like binding ofBacillus thuringiensis var.kurstaki lepidopteran-specific toxin is an initial step in insecticidal action. FEBS Lett. 168:197–202; 1984.
Knowles, B. P.; Francis, H.; Ellar, D. J. Structurally relatedBacillus thuringiensis δ-endotoxins display major differences in insecticidal activityin vivo andin vitro. J. Cell Sci. 84:221–236; 1986.
Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., et al. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275; 1951.
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65:55–63; 1983.
Schesser, J. H.; Kramer, K. J.; Bulla, L. A., Jr. Bioassay for homogenous parasporal crystal ofBacillus thuringiensis using the tobacco hornworm,Manduca sexta. Appl. Environ. Microbiol. 33:878–880; 1977.
Thomas, W. E.; Ellar, D. J. Mechanism of action ofBacillus thuringiensis var.israelensis insecticidal δ-endotoxin. FEBS Lett. 154:362–368; 1983.
Yamamoto, T.; Iizuka, T. Two types of entomocidal toxins in the parasporal crystals ofBacillus thuringiensis kurstaki. Arch. Biochem. Biophys. 227:233–241; 1983.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chow, E., Gill, S.S. A rapid colorimetric assay to evaluate the effects ofBacillus thuringiensis toxins on cultured insect cells. Journal of Tissue Culture Methods 12, 39–42 (1989). https://doi.org/10.1007/BF01578007
Issue Date:
DOI: https://doi.org/10.1007/BF01578007