A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

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Abstract
Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary.