Monitoring cytosolic calcium in parathyroid hormone target cells: Osteoblasts and renal epithelia

We describe methods for measuring cytosolic-free calcium concentrations [Ca²⁺]_i in cell monolayers, using the calcium-sensitive fluorescent probe fura-2. The system is based on a commercially available microspectrofluorometer, to which some additional hardware has been added to improve memory capability and time-resolution. Cells grown on glass cover slips can be observed and studied through an inverted microscope, coupled to the fluorometer. [Ca²⁺]_i can be monitored in real-time by either photon counting or video image analysis. These two techniques can be used selectively to take full advantage of their respective potentials: photon counting provides high sensitivity and time-resolution for experiments on single cells, whereas video imaging offers a spatial resolution of the calcium signal among the cell population and within single cells. The method can be applied to both primary cultures of dispersed proximal tubular cells as well as to immortalized or transformed cell lines.