RatioWorks™ BCFL, AM *Superior replacement for BCECF*

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 RatioWorks™ BCFL AM in high-quality, anhydrous DMSO.

   Note: When reconstituted in DMSO, RatioWorks™ BCFL AM is a clear, colorless solution.

1. On the day of the experiment, either dissolve RatioWorks™ BCFL AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a RatioWorks™ BCFL AM working solution of 5 to 50 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. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.

   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.

The following is a recommended protocol for loading RatioWorks™ BCFL AM into live mammalian cells. This protocol only provides a guideline and should be modified according to your specific requirements.

1. Prepare viable cells as desired.

2. On the next day, add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of the RatioWorks™ BCFL AM working solution into the cell plate.

   Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer (100 μL/well for 96-well plate or 25 μL/well for 384-well plate) before dye-loading.

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

6. Run the pH assay as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader at Ex/Em = 490/535 nm cutoff 515 nm. For ratio measurements, monitor fluorescence at Ex/Em₁ = 430/535 nm cutoff 515 nm and Ex/Em₂ = 505/535 nm cutoff 515 nm.

   Note: The compound addition is 50 μL/well (96-well plate) or 25 μL/well (384-well plate).

   Note: Assays should be completed within 3 to 5 minutes after compound addition. However, a minimum of 8 minutes is recommended for data collection.