Measurement by digital autoradiography with a beta-imager of [35S]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 cells were either treated with aracytosine or infected with an adenovirus encoding the Lac-Z gene according to two protocols of infection. In each experiment, 24 h before the end of the incubation period, [35S]methionine was added to one set of cultures which were performed in plastic chamber slides. At 10-13 days in vitro, control and treated cultures were processed either for immunocytochemical detection of neuron-specific enolase (NSE)-stained cells or for measurement of [35S]methionine incorporation. For the latter, cultures grown in the chamber slides were fixed with 4% paraformaldehyde, dehydrated, and air-dried. After removal of the upper structures of the chambers, the slides were directly transferred to a 1200 beta-imager, a gaseous detector which displays a digital image of the cultured cells and permits the quantitative measurement of incorporated [35S]methionine within a few hours. In aracytosine-treated cultures, we observed that the numbers of NSE(+) cells as well as [35S]methionine incorporation were decreased compared with control cultures. After viral infection, the number of NSE(+) neurons and the amount of radioactivity incorporated were either the same in control and infected cultures or decreased for the cultures treated according to the different protocols. In all cases, the amount of [35S]methionine incorporated varied in the same direction as the number 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 reliable marker of cell survival and/or proliferation.

Adenoviral-mediated transfection of the Lac-Z gene into rat dissociated embryonic central nervous system cells before and after seeding.

The adenovirus carrying a reporter gene--the Lac Z gene--is known to infect central nervous system (CNS) cells in primary cell cultures. The percentage of infected neurons with respect to the total number of neurons was studied in primary dissociated cultures as a function of the day of inoculation and the age of three rat CNS cultures: spinal cord, mesencephalon and cortex. Two methods of viral inoculation were compared: the first inoculation was performed on the cultured cell at 2, 3 or 6 days in vitro (DIV) whereas the second inoculation was performed on the cell suspensions before seeding. All the infected CNS cells has the same aspect as the control cultures. In the spinal cord and the mesencephalic cultures, the glial cells were preferentially infected, especially when the cells were inoculated at 6 DIV. In the cortical cultures, there were more infected neurons than infected glial cells. The number of CNS cells was lower when inoculation was performed at 6 DIV as compared with 3 DIV. Very few infected GABA cells were found in the cultures. A high percentage of infected neuronal cells relative to the total number of neuronal cells was found when infection of the three types of cultures was performed on the dissociated embryonic cell suspension before seeding.