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Modification of negative carboxlate by AM esters results in an uncharged, hydrophobic indicator or chelator that can permeate cell membranes.
Once inside the cell, intracellular esterases, found in almost all cell types, will hydrolyze the AM group. The resulting fluorescent indicator or chelator would then be contained inside the cell and accumulate.
What stock solutions should I make?
AM esters should be reconstituted using high-quality, anhydrous dimethylsulfoxide (DMSO). It is advisable to keep AM esters in as concentrated stock as possible (e.g. 1-10 mM) so that minimal amounts (ideally < 0.1%) of DMSO are present in the loading solution.
How do I store AM esters?
Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20°C, protected from light and kept anhydrous.
Solvents like DMSO will readily take up moisture, leading to decomposition of the dye.
Can I use AM esters in vitro?
This is intended as an introduction only. Specific protocols for any particular dye and cell
type should be obtained from the literature. Generally, AM esters are used at a final working concentration between 1-10 µM.
Incubation time typically ranges from 30 to 60 minutes. However incubation conditions can vary among cell types and among indicators and ideally should be optimized for each cell type.
It is best to use serum-free culture medium and incubate at room temperature or 37ºC (although loading is often better at room temperature).
Cells should be washed at least once with fresh serum-free culture medium to minimize extracellular background fluorescence. 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 (for example where MW >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.
Which surfactants and buffers should I use?
A surfactant 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 loading, the required volumes of the DMSO stock solutions of the AM ester and of the surfactant should be premixed and then dispersed into aqueous medium for loading.
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.