Improving techniques for clonogenic assays

A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.