RAPID, FLUOROMETRIC DNA DETERMINATION FOR CHICK LIMB-BUD MESENCHYMAL-CELL MICROCULTURES

Micromass cultures of chick and mouse limb-bud mesenchymal cells are c ommonly used for in vitro studies of cellular differentiation, Previou sly, adaptation of these cultures to 96-well plates facilitated analys es of various aspects of cellular behavior and the effects of differen t media components in these cultures. These adjustments allowed develo pment of a serum-free medium for cluck 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 3334 2-based in situ DNA assay that provides reliable data much more quickl y and with considerably less effort than had been feasible in the past . Because it allows quantitation of products of cellular differentiati on and DNA in the same cultures, the number of cultures needed to prov ide the same data is essentially halved and tile accuracy of normalize d values for quantitative estimated of markers of differentiation is i mproved. Studies of the effects of retinoic acid on chick limb-bud mes enchymal cells were performed to document the usefulness of this metho d.