Protonex™ Red 670 AM *Cell-Permeable*

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 10 to 20 mM stock solution of Protonex™ Red 670 AM in high-quality anhydrous DMSO.

   Note: An unused Protonex™ Red 670 AM stock solution should be divided into single-use aliquots and stored at ≤ -20 º C.  Protect from light and avoid repeated freeze-thaw cycles.

1. On the day of the experiment, either dissolve Protonex™ Red 670 AM dye in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

2. Prepare a Protonex™ Red 670 AM dye working solution of 5 to 20 μM in a buffer of your choice (e.g., Hanks and Hepes buffer).

   Note: The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. The final Pluronic® F-127 concentration should be approximately 0.02% in the staining buffer. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest (here). Avoid long-term storage of AM esters in the presence of Pluronic® F-127.

   Note: If your cells contain organic anion-transporters, probenecid (1-4 mM) may be added to the dye working solution (final in well concentration will be 0.5-2 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 (here). 

The following is a recommended protocol for loading Protonex™ Red 670 AM dye into live mammalian cells. This protocol only provides a guideline and should be modified according to your specific needs.

1. Prepare viable cells as desired (e.g., 100 µL/well/96-well plate or 25 µL/well/384-well plate).

2. On the next day, add the Protonex™ Red 670 AM dye working solution into the cell plate in equal volumes, such as 100 µL/well/96-well plate or 25 µL/well/384-well plate.

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

4. Replace the dye working solution with HHBS or buffer of your choice to remove any excess dye.

5. Prepare the compound plates using HHBS or a buffer of your choice.

6. Perform the pH assay as desired while simultaneously monitoring fluorescence. This can be done with either a fluorescence microscope that has a Cy5 filter set or a fluorescence plate reader at Ex/Em = 640/680 nm (cutoff 665 nm).