Gamma radiation (0.9-8.0 Gy) was used as a perturbing agent to study factors influencing in vitro chondrogenesis of embryonic chick limb bud cell culture. Chondrogenesis was measured using a number of criteria, including (1) cartilage nodule production, (2) spectrophotometric determination of the amount of bound Alcian blue dye, and (3) computer-assisted analysis of the spatial distribution (area) and density of Alcian blue present in individual micromass colonies. Gamma radiation inhibited both cell proliferation and chondrogenesis in a dose- and time-dependent fashion. Administration of benzamide caused a significant increase in cell proliferation at 0.9 and 2.7 Gy, and in chondrogenesis at all doses. Cartilage nodule production was affected during the first 2 days (prior to 48 h) of culture only, suggesting that chondrocytic commitment occurs during this period. Cultures irradiated at 48 and 72 h produced the same number of nodules as controls, but bound significantly less dye, presumably because of decreased cell numbers and/or cell synthesis products. Computer analysis of micromass colonies provided data similar to those collected spectrophotometrically, but displayed the advantages of (1) increased sensitivity to individual variations, (2) the ability to collect data sets without having to pool three or more colonies, and (3) long-term storage of raw images for later analysis.

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