Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Fura-2 AM diffuses across the cell membrane and is de-esterified by cellular esterases to yield Fura-2 free acid.
1. First, prepare the 1 mM Fura-2 AM stock by adding 50 μL of DMSO to a 50 μg vial. It is important to use dry DMSO packed under nitrogen and it is necessary to remove the DMSO with a needle by puncturing the septum to prevent hydration of the DMSO. After preparing the Fura-2 AM solution keep it in a dark dry place. Fura-2 AM in DMSO is stable at RT for 24 hours and is stable at -20 degrees in a dry container for several months.
2. Aliquot 2 mL of culture media into a 15 mL conical tube, warm to 37 deg. and add 2 μL of Fura-2 AM stock to generate a 1μM Fura-2 AM solution. Vortex the solution vigorously for 1 min.
3. Transfer the loading solution to a 35 mm tissue culture dish and transfer the coverslip with the cells into the dish.
4. Incubate the neurons at 37 degrees for 30 minutes in a dark incubator. Time the incubation precisely.
5. Prepare a 35 mm dish containing 2 mL of tissue culture media without Fura-2 AM. Remove the coverslip from the loading solution and place in the new dish.
6. Mount the coverslip on the imaging chamber.