A microwell assay for anchorage independent cell growth

Abstract

We describe modifications of the conventional assay for anchorage independent growth of fibroblasts that enable the assay to be carried out in microwell plates, as opposed to the conventional Petri dishes. The microwell assay is a good discriminator of final EGF concentrations in the range 10–100 picograms/ml, and can be used to detect absolute amounts of EGF below 2 pg. Addition of TGFβ to EGF enhances colony formation in the microassay in the usual manner. We describe the use of this microassay to identify and map local production of transforming growth factor activity by the component pieces of individual chick embryos. Transforming activity was identified in all the stages tested (Hamburger and Hamilton stages 13–23). Highest levels were found near the mid-line of the embryo. No clear differences in the cranio-caudal axis have so far been identified. This technique will enable the spatial and temporal distribution of transforming activity throughout vertebrate embryos to be completely mapped. It seems likely that this mapping process will help elucidate the normal role of transforming growth factors in embryos.