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PREPARATION OF WORKING SOLUTION：Fluo-2 AM Working Solution

On the day of the experiment, either dissolve Fluo-2 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, Fluo-2 AM at a final concentration of 4-5 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 Fluo-2 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. 

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