Method for combined 3H and 14C autoradiography with a single emulsion tested in cultured mammalian cells

A single-gelatin expanded film method for double-isotope autoradiography is described. A preliminary classification based upon silver-grain distribution is used to assign labeled cells to 3H only or with 14C classes. Optical sectioning combined with grain counting is employed to obtain ratios for classifying cells labeled with 14C into 14C only and 3H + 14C classes. The method has been tested with CHO-K1 cells in plateau-phase cultures using two 24-h labeling periods. The experimental design allowed for independent estimation of the expected frequencies of label classes under conditions that provided a wide range of possible label levels and combinations. Previous methods have used time-consuming applications of two emulsion layers and exposures to distinguish between cells labeled with 3H only or with 14C and do not identify the 3H + 14C class. A single-gelatin expanded film requires only one exposure and permits all label classes to be determined by an objective grain-counting procedure