Monitoring intracellular calcium using fluorescent dyes in a mid-throughput assay
Changes in the intracellular concentration of Ca2+ ions is the basis for numerous cellular responses; from receptor signaling to mediating contractile function. Thus accurate techniques to monitor intracellular [Ca2+] are in high demand for both academic and industrial research. Traditionally, monitoring intracellular Ca2+ requires live cell fluorescence imaging. Advances in microplate reader technology, including the ability to incubate at 37°C and 5% CO2 and inject reagents automatically, allow the adaptation of the assays to a microplate format which increases the throughput and automation capabilities.
Fluorescent dyes are used to monitor intracellular Ca2+ levels in living cells by fluorescent imaging. Here we have adapted such protocols for use in a 96-well plate format in the CLARIOstar® Plus plate reader. Upon histamine stimulation of HUVECs, Fura-2 and Cal-520AM displayed a comparable, high response to the induced intracellular Ca2+ changes. Dye leakage out of the cell can contribute significantly to the accuracy of intracellular Ca2+ measurements. We show that Cal-520AM remained in the cells over 30 min making it suitable for mid/high throughput analysis of intracellular Ca2+ levels in living cells.