Loading Fluorescent Ca2+ Indicators into Living Cells

Small-molecule fluorescent Ca(2+) reporters are the most widely used tools in the field of Ca(2+) signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca(2+) as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca(2+) indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca(2+) indicators have carboxylic acid groups for binding of Ca(2+). Because these "free-acid" forms of the indicators are hydrophilic they cannot readily cross cell membranes and need to be introduced into cells using techniques such as microinjection, pinocytosis, or diffusion from a patch pipette. However, the most convenient and widely used method for loading indicators into cells is as hydrophobic compounds in which the carboxylic acid groups are esterified (commonly as acetoxymethyl [AM] or acetate esters). The ester versions of the indicators permeate the plasma membrane. The Ca(2+)-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases.