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].

Instructions
I. Solution preparation
1. Preparation of mother solution: Mag-Fluo-4 AM is dissolved in DMSO to prepare a 10 mM mother solution.
2. Preparation of working solution: Dilute the mother solution with HBSS/PBS/H2O to prepare a 1-10 μM Mag-Fluo-4 AM working solution.
Notes:
It is recommended that the Mag-Fluo-4 AM mother solution be stored at -20℃ or -80℃ in the dark after aliquoting. Please avoid repeated freezing and thawing. Please adjust the concentration of Mag-Fluo-4 AM working solution according to actual conditions and prepare it before use.
II. Cell staining
1. Suspended cells
1) Collect cells by centrifugation and wash twice with PBS for 5 minutes each time. The cell density is 1×10^6/mL
2) Add 1 mL Mag-Fluo-4 AM working solution and incubate at room temperature for 5-30 minutes.
3) Set the centrifuge to 3400 g, centrifuge for 3-4 minutes, and discard the supernatant.
4) Add PBS to wash the cells twice, 5 minutes each time.
5) Resuspend the cells in 1 mL HBSS and observe using a fluorescence microscope or flow cytometer. (Excitation/emission wavelength reference: 480±15nm/535±30nm)[1]
2. Adherent cells
1) Culture the adherent cells on a sterile coverslip.
2) Remove the coverslip from the culture medium and remove excess culture medium.
3) Add 100 μL of Mag-Fluo-4 AM working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
4) Aspirate the Mag-Fluo-4 AM working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope.