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Loading fluorescent Ca2+ indicators into living cells.

Martin D Bootman¹, Katja Rietdorf, Tony Collins

¹Babraham Institute, Babraham, Cambridge, CB22 3AT, United Kingdom. martin.bootman@babraham.ac.uk

Cold Spring Harbor Protocols

|February 5, 2013

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Small-molecule fluorescent calcium (Ca2+) reporters are vital for cell signaling research. Understanding how to properly load these indicators into cells is crucial for obtaining accurate experimental data.

Area of Science:

Biochemistry
Cell Biology
Biophysics

Background:

Small-molecule fluorescent calcium (Ca2+) reporters are essential tools for studying Ca2+ signaling.
These reporters offer high spatial and temporal resolution, advancing our understanding of Ca2+ as a cellular messenger.
While often straightforward, loading these indicators into cells presents challenges for optimal data acquisition.

Purpose of the Study:

To highlight common pitfalls in the application of fluorescent Ca2+ indicators.
To provide guidance on best practices for cellular loading and monitoring of Ca2+ indicators.
To explain the chemical properties of Ca2+ indicators that influence their cellular uptake and activation.

Main Methods:

Discussion of the chemical properties of fluorescent Ca2+ indicators, focusing on their carboxylic acid groups.

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Explanation of the challenges associated with introducing hydrophilic, free-acid forms of indicators into cells.

Description of the widely used method of employing hydrophobic esterified forms (e.g., AM esters) for cellular loading.

Main Results:

Hydrophilic free-acid Ca2+ indicators require specialized delivery methods (microinjection, pinocytosis, patch pipette diffusion).
Hydrophobic esterified Ca2+ indicators readily cross cell membranes.
Intracellular esterases hydrolyze esterified indicators to release the active, Ca2+-sensitive free-acid form within the cell.

Conclusions:

The choice of indicator form (free-acid vs. esterified) significantly impacts cellular loading strategies.
Esterified fluorescent Ca2+ indicators offer a convenient method for intracellular delivery.
Proper loading techniques are critical for reliable Ca2+ signaling measurements using fluorescent reporters.