Advantages of Calcium Green-1 over Other Fluorescent Dyes in Measuring Cytosolic Calcium in Platelets

Abstract

Fluorescent indicators are widely used in the measurements of cytosolic calcium in many cell types for many purposes because they are relatively easy to use. Notwithstanding, they have some defects to prevent accurate measurements under certain conditions, such as significant dye leakage and UV-quenching effect. Menadione, a representative quinone derivative with antiaggregating effect, is also UV-absorbent. To investigate whether menadione can affect the change of cytosolic calcium in platelets by agonist, we measured the change of cytosolic calcium level using calcium green-1. Since this dye has not been used previously in platelets, we determined that the optimal loading of calcium green-1 to platelets was achieved using 3 μM dye incubated for 60 min at 37°C. Our study compared the use of calcium green-1 with fura-2 and fluo-3 (two widely used dyes) in measurements of cytosolic calcium. Fura-2 is UV-excited, so when menadione was treated in fura-2-loaded cells, it had a quenching effect. Fluo-3, the other visible fluorescent indicator, leaked from platelets very rapidly and required the use of anion channel blockers which are known to affect physiological response of platelets. Our study demonstrated that changes in cytosolic calcium levels can be accurately measured without these problems by using calcium green-1. We therefore were able to demonstrate that menadione inhibited calcium increase by thrombin in a dose-dependent manner similar to menadione's antiaggregating effect in platelets.