Tritium autoradiography of cell surfaces in smear preparations

Experiments were performed in order to find out whether tritium-labeled cell surface markers can be quantified at the single cell level in autoradiographs of smear preparations. Mouse thymocytes were incubated with 3H-concanavalin A and subsequently spread on microscopic slides. The spreading techniques, either by cytocentrifugation or by the use of cover slips, were performed in such a way as to achieve preparations in which the mean flatness of the cells varied. By means of incident light microphotometry, the cellular areas and the grain counts of individual cells were determined. The results show a strong dependence of the mean grain yield per slide on the mean cellular area. Cytocentrifuge preparations resulted in larger mean cellular areas and higher mean grain counts than cover slip preparations. With the use of cytocentrifugation, however, the differences in the flatness of the cells were of such a magnitude that a reproducible quantification of 3H-labeled cell surface markers was not possible. Conventional techniques of spreading cells on slides failed to provide a degree of flatness that could approach the saturation grain count per cell without completely destroying the cellular morphology.