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

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

1. 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 (20^o C) ~0 µM
   • 10 mM Ca²⁺ + 10mM EGTA in 100 mM KCl and 30 mM MOPS, pH 7.2:  Free Ca²⁺ in this solution (20^o C) ~39 µM 

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