General loading guidelines for AM esters
When cells are incubated with the AM ester of an indicator, the AM ester permeates into the cell and is hydrolyzed by intracellular esterases to yield the active form of the indicator, which becomes trapped and accumulated in the cell.
For loading, a 1-10 mM stock solution of the AM ester is prepared using anhydrous dimethylsulfoxide (DMSO) and stored at -20ºC. In order to prevent the deterioration of the AM esters that occurs with repeated thawing and freezing, we recommend that 1 mg or 500 µg quantities be divided into aliquots containing 50 µg each.
Loading is usually performed in a serum-free culture medium with the AM ester at a final concentration ranging from 1-10 µM. Pluronic F-127 may be added to the loading medium to aid dispersal of the AM esters; it is typically used at a concentration of < 0.1% (wt/vol). For ease of use, a stock solution of Pluronic F-127 should be made in dry DMSO at a concentration of 15% (wt/vol). For loading, the required volumes of the DMSO stock solutions of the AM ester and of the Pluronic should be premixed and then dispersed into aqueous medium for loading. The cells can be incubated at room temperature or 37ºC (although quality of loading is often better at room temperature), and the time of incubation typically ranges from 30 to 60 minutes. After loading, the cells should be washed at least once with fresh serum-free culture medium to minimize extracellular background fluorescence.
- If the loading medium is buffered with bicarbonate, then loading should be done under a 5% CO2 atmosphere to prevent alkalinization of the medium through loss of CO2;
- if serum-containing medium is used for loading, then the loading concentration of AM ester may need to be increased to compensate for binding of AM esters to serum proteins;
- in some cases (particularly when the AM ester has a molecular weight near or exceeding 1,000), the loaded cells may require a further incubation in medium without AM ester for 20 – 60 minutes to allow complete processing of the AM ester by intracellular esterases; and
- incubation conditions can vary among cell types and among indicators and ideally should be optimized for each cell type.