MEASUREMENT BY DIGITAL AUTORADIOGRAPHY WITH A BETA-IMAGER OF [S-35] METHIONINE INCORPORATED BY RAT CENTRAL-NERVOUS-SYSTEM CELLS IN PRIMARY CULTURES - A MARKER OF IN-VITRO DEVELOPMENT

Mixed glial-neuronal cultures prepared from rat embryonic cortical cel ls were either treated with aracytosine or infected with an adenovirus encoding the Lac-Z gene according to two protocols of infection. In e ach experiment, 24 h before the end of the incubation period, [S-35]me thionine was added to one set of cultures which were performed in plas tic chamber slides. At 10-13 days in vitro, control and treated cultur es were processed either for immunocytochemical detection of neuron-sp ecific enolase (NSE)-stained cells or for measurement of [S-35]methion ine incorporation. For the latter, cultures grown in the chamber slide s were fixed with 4%, paraformaldehyde, dehydrated, and air-dried. Aft er removal of the upper structures of the chambers, the slides were di rectly transferred to a 1200 beta-imager, a gaseous detector which dis plays a digital image of the cultured cells and permits the quantitati ve measurement of incorporated [S-35]methionine within a few hours. In aracytosine-treated cultures, we observed that the numbers of NSE(+) cells as well as [S-35]methionine incorporation were decreased compare d with control cultures. After viral infection, the number of NSE(+) n eurons and the amount of radioactivity incorporated were either the sa me in control and infected cultures or decreased for the cultures trea ted according to the different protocols. In all cases, the amount of [S-35]methionine incorporated varied in the same direction as the numb er of NSE(+) neurons in cultures. The digital imaging of the cultures permitted observation of the layer of cultured cells. It appears that such a rapid and direct measurement of incorporation of a radiolabeled indicator of protein synthesis may be considered as a quick and relia ble marker of cell survival and/or proliferation.