An assay for growth of mouse bone marrow cells in microtiter liquid culture using the tetrazolium salt MTT, and its application to studies of myelopoiesis

Abstract

Mouse bone marrow cells were grown in liquid culture in microtiter plates in the presence of different colony-stimulating factors (CSF). Growth was assayed using the tetrazolium salt MTT, which is reduced in the mitochondria of viable cells to a water-insoluble blue formazan dye. Two technical problems have limited the use of this assay: the solubilization of the dye crystals and the necessity to acidify the phenol red in the culture medium. Both could be solved here by the use of a developing solution of 5% formic acid in isopropanol. Using manual mixing combined with a short sonication by floating the plates in a sonic bath, the crystals were dissolved within minutes. There was no flocculation of protein, even using medium with 20% serum. The color remained stable for at least 4 h. This enabled the semi-automatic measurement of large numbers of cultures directly in the microtiter plates. Growth and differentiation of myelopoietic precursor cells in the liquid cultures was shown to be comparable to that in soft agar. Cell growth was CSF-dependent. The calculated cell yield per colony forming cell (CFC) seeded was within the range of the average cell number per colony found in soft agar, and the spectrum of mature cells obtained reflected the type of CSF used as stimulus. Using the combined culture and assay systems, it was possible to perform detailed kinetic studies of myelopoiesis. This technique should be useful for studying the mechanisms of action of pharmacological modulators of myelopoiesis.