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Loading fluorescent Ca2+ indicators into living cells

Bootman, Martin D.; Rietdorf, Katja; Collins, Tony; Walker, Simon and Sanderson, Michael (2013). Loading fluorescent Ca2+ indicators into living cells. Cold Spring Harbor Protocols, 2013(2), article no. pdb.prot072801.

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Small-molecule fluorescent Ca2+ reporters are the most widely used tools in the field of Ca2+ signaling. The excellent spatial and temporal resolution afforded by fluorescent reporters has driven the understanding of Ca2+ as a messenger in many different cell types. In many situations, the cellular loading and monitoring of fluorescent Ca2+ indicators is quite trivial. However, there are numerous pitfalls that require consideration to ensure that optimal data are recorded. Fluorescent Ca2+ indicators have carboxylic acid groups for binding of Ca2+. 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 Ca2+-sensitive, free-acid form of the indicator is liberated following hydrolysis of the ester groups by intracellular esterases.

Item Type: Journal Item
Copyright Holders: 2013 Cold Spring Harbor Laboratory Press
ISSN: 1559-6095
Academic Unit/School: Faculty of Science, Technology, Engineering and Mathematics (STEM) > Life, Health and Chemical Sciences
Faculty of Science, Technology, Engineering and Mathematics (STEM)
Item ID: 36742
Depositing User: Martin Bootman
Date Deposited: 01 Mar 2013 09:26
Last Modified: 08 Dec 2018 20:42
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