Fura-2 AM

Guideline (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific requirements).
Preparation of Stock Solution
Add anhydrous DMSO to Fura-2 AM, pipette gently or vortex until completely dissolved to prepare a 1 mM stock solution.
Cell Loading
2.1 Preparation of Working Solution (prepare freshly before use):
Preheat serum-free or low-serum medium to 37℃, add the above 1 mM stock solution, and mix by gentle inversion to prepare a 1 μM loading working solution.
(Optional) To facilitate uniform intracellular dye entry, 0.02% Pluronic F-127 or 0.1% BSA can be added.
2.2 Loading Incubation:
Transfer the coverslips with neuronal cells to a 35 mm culture dish containing the working solution.
Incubate at 37℃ for 30 minutes in the dark.
2.3 Post-incubation (De-esterification):
Preheat 2 mL of fresh dye-free culture medium.
Take out the coverslips from the loading solution and transfer them into the fresh medium.
Continue incubation at 37℃ for 20–30 minutes in the dark. This step is critical to remove extracellular residual dye and ensure sufficient intracellular hydrolysis of the dye into free Fura-2 acid.
3. Pre-imaging Preparation
3.1 Washing: Gently rinse the coverslips 1–2 times with prewarmed HBSS or imaging buffer (containing Ca2?/Mg2?) to eliminate background dye.
3.2 Mounting: Mount the coverslip onto the imaging chamber and add 1–2 mL of imaging buffer.
3.3 Stabilization: Place the chamber on a thermostatted microscope stage (37℃), equilibrate for 5–10 minutes before baseline recording.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.