Colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar

Abstract

In vitro colony forming unit (CFU) assays have been used to measure the effects of compounds that regulate the growth of hematopoietic progenitor cells. These assays are time consuming and subjective and are therefore not amenable to high throughput of large numbers of compounds. Here we have shown that the traditional murine bone marrow CFU assay can be modified into a robust non-subjective colorimetric assay format. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added after colony formation in an agar based 96-well plate culture system. Optical density correlated with increasing cell input concentrations in the presence of growth factor. The linearity of this response was equivalent to the standard CFU assay. Several hematopoietic inhibitors were tested in both assays. Effects on colony number and size were compared to optical density. Compounds that reduced colony numbers with little effect on colony size had identical IC 50 values in both the colorimetric assay and CFU assay. The IC 50 values of compounds that also decreased colony size did not correlate in the two assays. These results demonstrate the utility of the colorimetric assay to rapidly screen for compounds that specifically inhibit hematopoietic progenitor cell colony formation in vitro.