Prepare a 1 to 10 mM stock solution of AM esters just before use in high quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions should be stored well sealed, kept desiccated at - 20°C and protected from light. Under these conditions, AM esters should be stable for several months.

The nonionic detergent Pluronic^® F-127 is sometimes used to increase the aqueous solubility of AM esters.

Note: An equal volume of 20% Pluronic^® F-127 solution can be added to DMSO stock solutions before diluting into the loading buffer. The final Pluronic^® F-127 concentration is about 0.02%. A variety of Pluronic^® F-127 solutions can be purchased from AAT Bioquest. The long-term storage of AM esters in the presence of Pluronic^® F-127 is not recommended.  

On the day of the experiment, either dissolve AM esters in DMSO or thaw an aliquot of the indicator stock solutions to room temperature. Prepare a working solution of 1 to 10 µM (higher concentrations of weakly fluorescent indicators may be required) in the buffer of your choice (such as Hanks and Hepes buffer). For most of cell lines, AM esters (4-5 µM) are recommended. The exact concentration of indicators required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.

If the cells containing the organic anion-transports, probenecid (1-2.5 mM) or sulfinpyrazone (0.1- 0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators. Incubate cells with the AM esters for 20 minutes to one hour (Longer incubation time is required for weakly fluorescent indicators) at room temperature or 37°C.

Wash cells in dye-free buffers (containing an anion transporter inhibitor, if applicable) to remove excess probes.

Measure fluorescence intensity at desired Ex/Em wavelengths.