A Highly Selective Green Fluorescent Magnesium Indicator for Intracellular Magnesium Ion Analysis

Abstract

Intracellular magnesium ion is a crucial ion for meditating enzymatic reactions, DNA synthesis, hormonal secretion and muscle contractions. There are a few commercial fluorescent indicators used for the measurement of Magnesium ion in cells. However, all the commercially available indicators are based on tricarboxylate APTRA chelator derived from the popular tetracarboxylate BAPTA calcium ion chelator, including mag-fura-2, mag-indo-1, mag-fluo-4 and mag-rhod-2. Measurement of magnesium ion using these fluorescent indicators is challenging since mag-fura-2, mag-indo-1, mag-fluo-4 and mag-rhod-2 have higher affinity to calcium than magnesium. Magnesium and calcium ion-induced cellular signals are often intertwined, thus the poor selectivity of the existing indicators caused severe artifacts and interferences by calcium ion-induced signals. In addition, the physiological changes in magnesiumconcentrations are generally not as great as calcium concentration change upon biological stimulations, thus demanding more sensitive and selective magnesium indicators. Mag-520 has been developed to address the drawbacks of the existing magnesium ion indicators. We demonstrated that Mag-520 can detect the magnesium from 0.1 to10 mM. It has 5 times higher selectivity for magnesium than calcium while mag-fluo-4 has higher affinity to calcium than magnesium. Mag-520, AM was further evaluated for intracellular changes in magnesium using several cell lines such as HL-60, HEK-293, and HeLa cells with microplate reader, flow cytometry and fluorescence microscope. The intracellular magnesium ion level changes from forskolin stimulation can be readily detected with Mag-520, AM. In conclusion, Mag-520 is a highly selective magnesium ion indicator and can be applied in the detection of cellular Magnesium ion influx with very low interference from Calcium ion signaling. Mag-520 can be easily used with widely available FITC filter set. Compare to other magnesium ion detection dyes, Mag-520 has better performance inside the cells.