Fura Red, AM *CAS 149732-62-7*

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. Prepare a 2 to 5 mM stock solution of Fura Red AM in high-quality, anhydrous DMSO.

1. On the day of the experiment, either dissolve Fura Red AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

2. Prepare a 2 to 20 µM Fura Red AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Fura Red AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.

   Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Fura Red AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.

   Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.

Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.

1. Prepare cells in growth medium overnight.

2. On the next day, add 1X Fura Red AM working solution to your cell plate.

   Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.

3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.

   Note: Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.

4. Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
5. Add the stimulant as desired and simultaneously monitor fluorescence intensity using a fluorescence plate reader, which contains a programmable liquid handling system such as a FlexStation, at Ex/Em₁ = 435/630 nm cutoff 610 nm and Ex/Em₂ = 470/650 nm cutoff 630 nm.