A simplified method for determination of tritiated thymidine incorporation into cells from tissue in soft agar culture.

We designed a simple method for in vitro chemosensitivity testing of tritiated thymidine incorporation by cells from tumor tissue plated in soft agar. Cells from tumor tissue were cultured in a plastic dish, of which a Millipore membrane had been sealed to the underside, containing two layers of agar. After labeling the cells with tritiated thymidine, the dishes were placed in a boiling water bath to solubilize the agar. The labeled cells could be readily collected on the membrane by evacuating the solubilized medium through membrane filters sealed underside of the dish. Using this system, 11 (55 per cent) of 20 tumor specimens from 20 patients with various carcinomas showed an incorporation of over 200 dpm above background and 80 per cent or greater inhibition of thymidine uptake by sodium azide treatment. This method offered several advantages over the thymidine incorporation assay, including technical simplicity and a shorter time course.