How do you measure Ca2+ response with calcium indicators like Calbryte™ 520, potassium salt?

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Below is a sample protocol for measuring calcium response using Calbryte™ 520, potassium salt (Cat# 20658).

Begin by creating 10-15 µM probe solution in the following buffer:

10 mM K2EGTA in 100 mM KCl and 30 mM MOPS, pH 7.2:  Free Ca2+ in this solution (20o C) ~0 µM
10 mM Ca2+ + 10mM EGTA in 100 mM KCl and 30 mM MOPS, pH 7.2:  Free Ca2+ in this solution (20o C) ~39 µM

Measure the fluorescence change at Ex/Em= 490/525 nm (cutoff=515 nm) with a microplate reader or fluorescence spectrometer.
Calculate the fold of intensity increase.

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