Abstract. We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb‐bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage‐17 and ‐19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb‐bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non‐chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb‐bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.
A new method has been developed for culturing 11-day mouse forelimb buds in vitro. In cultures performed with conventional procedures, skeletal pieces frequently appeared distorted and reduced in size. Moreover, forelimb buds explanted from embryos younger than a stage corresponding to 50 pairs of somites developed narrow hand plates devoid of radiated autopods. By contrast, in the new procedure using media supplemented with fetal calf serum and growth factors and enhancing distal feeding with carrier implants of catgut, enlarged pads were obtained that exhibited at least 4 digital rays in buds explanted from embryos with 40-44 pairs of somites. Compared with conventional procedures, the mean value of DNA content per limb bud was twice as great with use of our improved method. The ability of limb bud cells to proliferate and differentiate when cultured either in classical or in modified conditions, and the importance of the technical procedures, are discussed in the new prospect of in vitro developmental studies.
An enzyme-linked immunosorbent assay (ELISA) was applied to a light protein, isolated from human breast cyst fluid (BCF) termed "gross cystic disease fluid protein - 15 Kda" (GCDFP-15), a potential differentiation marker in in vitro human breast cancer studies. The detection limits of this procedure, performed in microtiter plates, were 0.5 to 250 ng/well corresponding to 10 ng/ml to 5 micrograms/ml of sample or antigen solution. Possible cross-reaction with various antigens, especially those found in culture media, were investigated. The correlation coefficient between enzymoassay and radioimmunoassay was 0.978. The results showed that quantification of GCDFP-15 by ELISA is a specific and highly sensitive method. This procedure may be of interest in in vitro studies on the functional differentiation of breast cancer cells.
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