Protocol for monitoring cytoplasmic Ca2+ dynamics using Fura Red-AM on a Zeiss Axio Observer microscope

Here, we present a protocol for live-cell calcium imaging in HEK, HeLa, fibroblast, and myoblast cells using Fura Red-AM and a Zeiss Axio Observer inverted microscope. We describe steps for dye preparation and loading, microscope setup, dual-excitation image acquisition, and ratiometric signal analysis. The setup is optimized for store-operated calcium entry (SOCE), based on the activation of STIM1 and ORAI1. This protocol enables real-time monitoring of intracellular calcium dynamics and supports both non-transfected and GFP-transfected cells. • Instructions for calcium imaging using Fura Red on a Zeiss Axio Observer microscope • Steps for dye loading, image acquisition, and dual-wavelength ratio analysis • Setup guidance for ZEN software to record calcium flux in single live cells • Protocol enables real-time SOCE measurement in multiple adherent cell types Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for live-cell calcium imaging in HEK, HeLa, fibroblast, and myoblast cells using Fura Red-AM and a Zeiss Axio Observer inverted microscope. We describe steps for dye preparation and loading, microscope setup, dual-excitation image acquisition, and ratiometric signal analysis. The setup is optimized for store-operated calcium entry (SOCE), based on the activation of STIM1 and ORAI1. This protocol enables real-time monitoring of intracellular calcium dynamics and supports both non-transfected and GFP-transfected cells.