Rapid, fluorometric DNA determination for chick limb-bud mesenchymal-cell microcultures.
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.
Paulsen
D F
DF
Department of Anatomy, Morehouse School of Medicine, Atlanta, Georgia 30310-1495, USA.
Chen
W D
WD
Scineaux
T
T
Adams
D
D
D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D018878:Limb Buds / Q000378:metabolism*; D008648:Mesoderm / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism; D013997:Time Factors
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride