Fluorescence spectrophotometer for the real time detection of cytosolic free calcium from cell suspensions during exposure to extremely low frequency magnetic fields

Abstract

We have previously observed that cytosolic free calcium ([Ca2+]i) is increased in HL-60 cells after exposure to an extremely low frequency (ELF) magnetic field. To facilitate the investigation of the time dependence of this effect, we built a magnetic field exposure system in which four samples of cells could be kept at constant temperature and maintained in suspension with constant mixing. Fluorimetric measurements of [Ca2+]i from these samples were made in real time using a periscopic optical system attached to a fluorescence spectrophotometer. This design feature eliminated possible confounding influences of the applied magnetic field on the light detection electronics, and isolated the cell samples against stray magnetic fields from electronic equipment. The operation of the instrument was computerized. This permitted the precise definition and replication of experimental conditions such as the properties of the applied magnetic field, the rate of sample mixing, and the instant at which an agonist was added to the cells. It also provided a time-resolved record of sample temperature and fluorescence for each experiment. Characterization of the instrument enabled us to rule out potential confounding influences on fluorimetric [Ca2+]i measurements, such as temperature and mechanical vibration, and to have confidence in the accuracy and precision of the applied ELF and static magnetic fields. Experiments with HL-60 and Jurkat cells verified that the instrument could measure [Ca2+]i in live cells and follow large rapid changes in [Ca2+]i after activation with an agonist. This instrument will provide an ideal tool with which to investigate in greater detail the effect of ELF magnetic fields on [Ca2+]i in HL-60 cells.