Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart

Abstract

Stimulated by difficulties experienced when using fluo-3 AM, we developed a general mechanistic model to aid understanding and practical application of calcium probes applied as acetoxymethyl (AM) esters. Several practical issues previously overlooked or under-emphasized are considered by this model. First, some AM ester probes are “super” lipophilic, e.g., calcium orange, fluo-3, fura red, and these are trapped in the plasma membrane. Entry of such compounds into cells requires the presence of serum albumin in the incubation medium or esterase in the plasma membrane or both. Second, visible cytosolic calcium signals require significant cytosolic esterase, which varies considerably among cell lines and within cell populations of a single cell line. Finally, compartmentalization artefacts are most likely when incompletely hydrolyzed esters are present in the cytosol. This can occur because of low cytosolic esterase concentration or activity, and especially when long incubation times or high extracellular probe concentrations are used. An additional factor favoring compartmentalization is the presence of the “salt” form of the probe in the cytosol in the absence of significant concentrations of calcium ions. We provide an algorithmic chart to aid assessment of possible compartmentalization, guides to relevant QSAR models, and notes on estimation of the structural parameters required when using these models.

Key words::

• calcein
• calcium green-1
• calcium orange
• fluo-3
• fluorescent probe
• fura-2
• fura red
• indo-1
• QSAR model
• quin-2
• rhod-2

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Correction to: Predicting and avoiding subcellular compartmentalization artifacts arising from acetoxymethyl ester calcium imaging probes. The case of fluo-3 AM and a general account of the phenomenon including a problem avoidance chart.

Acknowledgments

R.W.H. thanks Dr. R. Aitken, School of Life Sciences, College of Medical, Veterinary and Life Science, Glasgow University, Scotland UK for providing facilities. K.T and P.D are indebted to a HEA PRTLI4 Grant to Fund the National Biophotonics and Imaging Platform Ireland (www.nbipireland.ie) and IRCSET Enterprise Partnership Scheme in conjunction with Hewlett-Packard Galway Ltd.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.