Replica plating and in situ enzymatic assay of animal cell colonies established on filter paper

Abstract

We have developed a simple technique for the replica plating of Chinese hamster ovary (CHO) cells. In this procedure cells are allowed to divide for 8-16 days between the plastic surface of a petri dish and a disc of Whatman no. 50 filter paper, weighted down with glass beads. The culture medium can be replaced when necessary without disturbing the growing colonies. Cells from each developing colony grow into the fibers of the paper, while others remain attached to the plate. The cell colonies transferred to the paper are viable and can be replica plated to a new petri dish with high resolution. In this way several inositol auxotrophs have been identified in a stock of mutagen treated cells without prior enrichment. Alternatively, the cells on the paper can be rendered permeable in situ, which permits autoradiographic screening for specific biochemical defects, as reported previously for Escherichia coli [Raetz, C.R.H. (1975) Proc. Natl. Acad. Sci. USA 72, 2274-2278]. This technique is applicable to other common cell lines and is especially useful for the identification of single colonies defective in the synthesis of DNA, RNA, protein, and membrane lipids.