Mag-Fluo-4 AM, a fluorescent Ca 2+ chelator, exhibits high affinity for calcium and is specifically designed to detect intracellular calcium ions. It features high sensitivity and low cytotoxicity. The compound's AM acetylmethyl ester form enhances cellular entry, and once inside, it is cleaved by intracellular esterases. This allows it to remain in the cell and bind to calcium ions, resulting in strong fluorescence [1].

Mag-Fluo-4 AM Working Solution Preparation: First, prepare a 10 mM stock solution by diluting Mag-Fluo-4 AM in anhydrous DMSO. Note that it is recommended to aliquot and store this stock solution at -20℃ or -80℃, protected from light. For working solution preparation, dilute the stock with pre-warmed HBSS to make a 1-10 μM solution, adjusting the concentration as needed and preparing fresh solutions for immediate use.

For cell staining of suspension cells: Collect the cells by centrifugation, wash twice with PBS for 5 minutes each, maintaining a density of 1×10⁶/mL. Add 1 mL of the Mag-Fluo-4 AM working solution and incubate at room temperature for 5-30 minutes. Centrifuge at 400 g for 3-4 minutes, remove the supernatant, and wash the cells twice with PBS for 5 minutes each. Resuspend the cells in 1 mL HBSS and observe using a fluorescence microscope or flow cytometer.

For cell staining of adherent cells: Culture adherent cells on a sterile coverslip. Remove the coverslip from the medium, aspirate excess medium, and add 100 μL of the dye working solution, ensuring it covers the cells entirely by gentle mixing. Incubate for 5-30 minutes, remove the dye solution, wash the cells 2-3 times with medium for 5 minutes each, and observe using a fluorescence microscope.