Mag-Fluo-4 AM is a Mg 2+ (magnesium ion) dye and a low-affinity Ca 2+ (calcium ion) dye and fluorescent indicator with Kd of 4.7 mM and 22 µM, respectively, that localizes Ca 2+ in the lumen of endoplasmic reticulum-like tubules in many cell types, and is commonly used in live cell assays.

1. Solution Preparation:
Stock Solution: Prepare a 2 to 5 mM Mag-Fluo-4 AM stock solution in high-quality anhydrous DMSO.
Working Solution: On the day of the experiment, dissolve Mag-Fluo-4 AM in DMSO, or thaw the stock solution to room temperature, and then prepare a 2 to 20 μM working solution of Mag-Fluo-4 AM in the chosen buffer (e.g., Hanks or Hepes buffer) with 0.04% Pluronic (8F-127). For most cell lines, the recommended final concentration of Mag-Fluo-4 AM is 4 to 5 μM.
2. Cell Preparation: Cultivate cells overnight in growth medium.
3. Dye Loading: The next day, add 1X Mag-Fluo-4 AM working solution to the cell culture plate. If the compound may interfere with serum, replace the growth medium with fresh HHBS buffer prior to dye loading.
4. Incubation: Incubate the dye-loaded culture plates in a 37°C cell culture incubator for 30 to 60 minutes.
5. Removal of Excess Probe: Replace the dye working solution with HHBS or the chosen buffer (optionally including an anion transporter inhibitor such as 1 mM probenecid) to remove excess probe.
6. Stimulation and Fluorescence Measurement**: Add stimuli as needed and measure fluorescence using a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader with a programmable liquid handling system, at Ex/Em = 490/525 nm with a 515 nm cutoff.