Summary
Micromass cultures of chick and mouse limb-bud mesenchymal cells are commonly used for in vitro studies of cellular differentiation. Previously, adaptation of these cultures to 96-well plates facilitated analyses of various aspects of cellular behavior and the effects of different media components in these cultures. These adjustments allowed development of a serum-free medium for chick limb-bud mesenchymal cells and substantially decreased costs associated with media and reagents. Here we report a further development for this model system; a Hoechst 33342-based in situ DNA assay that provides reliable data much more quickly and with considerably less effort than had been feasible in the past. Because it allows quantitation of products of cellular differentiation and DNA in the same cultures, the number of cultures needed to provide the same data is essentially halved and the accuracy of normalized values for quantitative estimates of markers of differentiation is improved. Studies of the effects of retinoic acid on chick limb-bud mesenchymal cells were performed to document the usefulness of this method.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ahrens, P. B.; Solursh, M.; Reiter, R. S. Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60:69–82; 1977.
Biddulph, D. M.; Sawyer, L. M.; Dozier, M. M. Chondrogenesis in chick limb mesenchyme in vitro derived from distal limb bud tips: changes in cyclic AMP and in prostaglandin responsiveness. J. Cell. Physiol. 136:81–87; 1988.
Brunk, C. F.; Jones, K. C.; James, T. W. Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem. 92:497–500; 1979.
Daniels, K.; Reiter, R.; Solursh, M. Micromass cultures of limb and other mesenchyme. In: Bronner-Fraser, M., ed. Methods in avian embryology. New York: Academic Press; 1996:237–247.
Finch, R. A.; Zwilling, E. Culture stability of morphogenetic properties of chick limb-bud mesoderm. J. Exp. Zool. 176:397–408; 1971.
Kapuscinski, J.; Skoczylas, B. Simple and rapid fluorimetric method for DNA microassay. Anal. Biochem. 83:252–257; 1977.
Labarca, C.; Paigen, K. A simple, rapid, and sensitive DNA assay. Anal. Biochem. 102:344–352; 1980.
Leonard, C. M.; Bergman, M.; Frenz, D. A., et al. Abnormal ambient glucose levels inhibit proteoglycan core protein gene expression and reduce proteoglycan accumulation during chondrogenesis: possible mechanism for teratogenic effects of maternal diabetes. Proc. Natl. Acad. Sci. USA 86:10113–10117; 1989.
Lev, R.; Spicer, S. S. Specific staining of sulfate groups with alcian blue at low pH. J. Histochem. Cytochem. 12:309; 1964.
Meyer, J. Ch.; Grundmann, H. Fluorometric determination of DNA in epidermis and cultured fibroblasts using 4′-6-diamidino-2-phenylindole (DAPI). Arch. Dermatol. Res. 276:52–56; 1984.
Nagata, Y.; Yokota, H.; Kosuda, O., et al. Quantification of picogram levels of specific DNA immobilized in microtiter wells. FEBS Lett. 183:379–382; 1985.
Otto, F.; Tsou, K. C. A comparative study of DAPI, DIPI and Hoechst 33258 and 33342 as chromosomal DNA stains. Stain Technol. 60:7–11; 1985.
Paulsen, D. F. Retinoic acid in limb-bud outgrowth: review and hypothesis. Anat. Embryol. 190:399–415; 1994.
Paulsen, D. F.; Chen, W.-D.; Okello, D., et al. Stage-and region-dependent responses of chick wing-bud mesenchymal cells to retinoic acid in serum-free microcultures. Dev. Dyn. 201:310–323; 1994.
Paulsen, D. F.; Chen, W.-D.; Pang, L., et al. Stage-and region-dependent chondrogenesis and growth of chick wing-bud mesenchyme in serumcontaining and defined tissue-culture media. Dev. Dyn. 200:39–52; 1994.
Paulsen, D. F.; Langille, R. M.; Dress, V., et al. Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid. Differentiation 39:123–130; 1988.
Paulsen, D. F.; Solursh, M. Microtiter micromass cultures of limb-bud mesenchymal cells. In Vitro Cell. Dev. Biol. 24:138–147; 1988.
Paulsen, D. F.; Solursh, M.; Langille, R. M., et al. Stable position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures. In Vitro Cell. Dev. Biol. 30A:181–186; 1994.
Plummer, H. K.; Dillard, C. Y.; Murray, S. A. Hoechst 33342 for microfluorometric measurement of adrenocortical tumor cell proliferation. In Vitro Cell. Dev. Biol. 28A:4–6; 1992.
Richards, W. L.; Song, M.-K.; Krutsch, H., et al. Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorometric determination of chromatin DNA. Exp. Cell Res. 159:235–246; 1985.
Summerbell, D.; Wolpert, L. Cell density and cell division in the early morphogenesis of the chick wing. Nat. New Biol. 239:24–26; 1972.
West, D. C.; Sattar, A.; Kumar, S. A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal. Biochem. 147:289–295; 1985.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Paulsen, D.F., Chen, WD., Scineaux, T. et al. Rapid, fluorometric DNA determination for chick limb-bud mesenchymal-cell microcultures. In Vitro Cell.Dev.Biol.-Animal 34, 158–162 (1998). https://doi.org/10.1007/s11626-998-0099-5
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11626-998-0099-5