SoNa™ 520 AM

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

Prepare a 2 to 5 mM stock solution of SoNa™ 520 AM in high-quality, anhydrous DMSO.

On the day of the experiment, either dissolve SoNa™ 520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, SoNa™ 520 at a final concentration of 5-10 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 SoNa™ 520 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 SoNa™ 520 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 1 to 2 hours.

   Note: Incubating the dye for longer than 2 hours 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 measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.