title: Loading fluorescent Ca2+ indicators into living cells.
abstract: 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.
authors: Babraham Institute, Babraham, Cambridge, CB22 3AT, United Kingdom. martin.bootman@babraham.ac.uk
authors: Martin D
authors: MD
authors: Bootman
authors: Katja
authors: K
authors: Rietdorf
authors: Tony
authors: T
authors: Collins
authors: Simon
authors: S
authors: Walker
authors: Michael
authors: M
authors: Sanderson
mesh_terms: D002118:Calcium / Q000008:administration & dosage* / Q000737:chemistry; D002264:Carboxylic Acids / Q000737:chemistry; D004058:Diffusion; D005456:Fluorescent Dyes / Q000008:administration & dosage* / Q000737:chemistry; D008845:Microinjections; D010873:Pinocytosis
chemical_list: D002264:Carboxylic Acids; D005456:Fluorescent Dyes; D002118:Calcium