I am planning to use Fura-2 AM to detect calcium levels in primary neurons, but the experimental methods in the references I searched for are somewhat different. In terms of reagent preparation, some protocols recommend dissolving Fura-2 AM in DMSO to prepare a stock solution, but the concentration varies from 1 mM to 5 mM. In addition, there are different opinions on which buffer to use when preparing the working solution, such as Hank's buffered saline solution (HBSS), HEPES-BSA buffer, or other buffers. When treating cells, there are also great differences in incubation time and temperature. Some teachers recommend incubating at 37°C for 15-60 minutes, while others recommend incubating at room temperature for 20 minutes. How do I accurately prepare the Fura-2 AM stock solution (including the amount of Fura-2 AM powder, the amount of DMSO, and any special mixing requirements)? What is the optimal working solution formulation (buffer type, additives (e.g., Pluronic F-127) and their concentrations)? Specific steps for cell treatment, including cell seeding density (if relevant), incubation time, temperature, and the proper method for washing cells before and after incubation with Fura-2 AM.
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 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.
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