Cal-520ER™ 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

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

   Note: When reconstituted in DMSO, Cal-520ER™ AM is a clear, colorless solution.

1. On the day of the experiment, either dissolve Cal-520ER™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

2. Prepare a 2 to 20 µM Cal-520ER™ AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127, and 1-2 mM of probenecid (final in well concentration will be 0.5-1mM). For most cell lines, Cal-520ER™ AM at a final concentration of 10-20 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 Cal-520ER™ AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.

   Note: If your cells contain organic anion-transporters, probenecid 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 Cal-520ER™ 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 2 to 3 hours.

   Note: The incubation time for specific cell lines may require optimization to improve signal intensities.

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.