Can I combine Fura Red with Fluo-3 or Fluo-4 for ratiometric analysis?

When Fura Red is combined with Fluo-3, the resulting ratiometric outcomes are comparable to using Indo-1. While Fura Red alone is also compatible with ratiometric analysis and has been utilized for detection via confocal microscopy, its use in real-time calcium flux measurements by flow cytometry is restricted. Fura Red exhibits reduced fluorescence when bound to calcium, providing a contrast to the behavior of Fluo-3/Fluo-4 that increases fluorescence upon binding calcium. By measuring the ratio of the signals from Fluo-3/Fluo-4 and Fura Red, researchers can obtain a ratiometric measurement of intracellular calcium levels.