Autoradiography of Soluble Materials

An autoradiographic method has been developed by which the presence of soluble intracellular materials can be determined for individual cells (1). Our immediate need for the method was to measure the uptake of H3-thymidine into soluble pools during various stages of the cell life cycle, but it should be applicable to the assay of various soluble radioactive materials in a variety of cell types under many experimental conditions. Some results of studies on the uptake of exogenous H 3-thymidine by the ciliate Tetrahymena and a mam-malian tissue culture cell are given to illustrate the method. For this technique, cells must be prepared for autoradiography by procedures which either prevent loss of soluble material from the cell or limit diffusion of soluble materials to a relatively small area surrounding the cell. Standard freeze-drying techniques are suitable for tissue sections, while both freeze-drying and air-drying can be used for tissue cultures. Most microorganisms can be prepared by rapid air-drying of individual cells or freeze-drying of wet smears on microscope slides. It is essential always that the cells be washed free of exogenous radioactivity before drying or freezing. To differentiate between soluble and insoluble radioactivity, samples of dried ceils are treated in the following ways before emulsion application. COMPLETE EXTRACTION OF SOLUBLE MATERIAL : Labeled cells mounted on slides are extracted x~ith acid alcohol, trichloroacetic acid, etc., according to the usual procedure. Auto-radiographs of such cells demonstrate the amount and location of insoluble (incorporated) radioactivity. Since the soluble materials have been removed, emulsions applied by standard liquid emulsion techniques or by the dry film technique described below give the same results. If cells have been prepared in a manner which does not retain soluble materials in the ceil but limits the area of diffusion, application of dry emulsion films permits the identification of individual cells from which soluble radioactivity has been derived.