A study of the fluorescence measurement using a 96-well microplate by a remodelled parallel luminescent measuring system.

Abstract

To develop an index to measure alveolar macrophage activity, the fluorescent technique for detection of calcium flux was paid special attention. In this study, a parallel luminescence measuring system was remodelled for fluorescence measurement using a 96-well microplate. The fluorescence indicators widely used to measure cytosolic free calcium ion concentration require excitation at ultraviolet (UV) wavelengths. Instruments to produce UV wavelengths are expensive compared to those used to produce visible wavelengths, and UV wavelengths are potentially injurious to cells. To avoid these problems, Fluo 3 (excitation wavelength in the visible range) was used as the fluorescent dye for detecting calcium ions. The parallel luminometer was remodelled successfully for fluorescent measurement as assessed by the results obtained from the measurements of a common fluorescent dye, fluorescein. Concentrations of free calcium ions were measured using Fluo 3 at 37 degrees C to consider the measurement of calcium flux in living cells. Although a linear relationship between concentrations of free calcium ions and fluorescence were observed, a diminution of fluorescence over time was also observed. To measure calcium flux in living cells, further instrumental and experimental improvements are thus needed.