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Monitoring intracellular calcium using fluorescent dyes in a mid-throughput assay

Thomas P. Keeley, Ron Jacob, Giovanni E. Mann, King’s BHF Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, London SE1 9NH, UK King’s BHF Centre of Research Excellence, School of Cardiovascular Medicine and Sciences, King’s College London 03/2019

Introduction

Changes in the intracellular concentration of Ca2+ ions is the basis for numerous cellular responses; from receptor signalling 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, have allowed the adaptation of the traditional fluorescence-based assays to a microplate format. This greatly increases the throughput and automation of such assays.


This application note compares the suitability of 3 commercially available fluorescent Ca2+ dyes; Fura-2AM, Fluo-8AM and Cal-520AM used to monitor histamine-stimulated Ca2+ mobilisation in human umbilical vein endothelial cells in the CLARIOstar® plate reader equipped with atmospheric control unit.


Assay Principle

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.

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