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.

This article refers 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

K Thompson, P Dockery, RW Horobin. 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. Biotechnic & Histochemistry 2012;87(7):468–483.

Spacing errors in the tables may interfere with their interpretation. The tables have been re-formatted and reproduced below. We regret any confusion that may have been caused by these errors.

Table 1. Key physicochemical properties and predicted intracellular localizations of fully, partially and totally de-esterified species of various widely used calcium probes

Probe and species^1	Z^2	Log P^3	Predicted membrane permeance^4 and accumulation sites^5	Comments
Calcein
Esterified	0	5.4	Permeant; biomembranes
Part de-esterified Least anionic Most anionic	1− 2−	3.1 −1.0	Lysosomes; mitochondria	Possible hydrolysis product; some may go to ER (see note 6)
De-esterified salt	6−	−12.0	Nuclei (histones)
Calcium green-1
Esterified	0	7.0	Permeant; biomembranes
Part de-esterified Least anionic Most anionic	1− 3−	2.3 −2.5	Lysosomes; mitochondria	One of several possible ester hydrolysis products
De-esterified salt	6−	−8.7	Nuclei (histones)
Calcium orange
Esterified	0	9.1	Impermeant; plasma membrane	A zwitterion
Part de-esterified Least anionic Most anionic	0 1−	7.5 0.4	Lysosomes; mitochondria	Possible hydrolysis product; some such may go to ER (see note 6)
De-esterified salt	4−	−2.5	Nuclei (histones)
Fluo-3
Esterified	0	11.2	Impermeant; plasma membrane
Part de-esterified Least anionic Most anionic	1− 4−	9.1 −0.3	Lysosomes; mitochondria	Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)
De-esterified salt	5−	−4.3	Nuclei (histones)
Fura-2
Esterified	0	7.0	Permeant
Part de-esterified Least anionic Most anionic	0 3−	5.6 −3.2	Lysosomes; mitochondria	Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)
De-esterified	5−	−9.0	Nuclei (histones)
Fura red
Esterified	0	10.3	Impermeant; plasma membrane
Part de-esterified Least anionic Most anionic	0 3−	5.5 −1.6	Lysosomes; mitochondria	Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)
De-esterified salt	4−	−4.4	Nuclei (histones)
Indo-1
Esterified	0	8.7	Impermeant; plasma membrane
Part de-esterified Least anionic Most anionic	0 3−	7.2 −1.6	Lysosomes; mitochondria	Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)
De-esterified salt	5−	−7.4	Nuclei (histones)
Quin-2
Esterified	0	6.4	Permeant; biomembranes
Part de-esterified Least anionic Most anionic	0 3−	4.8 −2.3	Lysosomes; mitochondria	Possible hydrolysis product; some may go to endoplasmic reticulum (see note 6)
De-esterified salt	4−	−5.2	Nuclei (histones)
Rhod-2
Esterified	1+	5.8	Permeant; mitochondria
Part de-esterified Least anionic Most anionic	1 + 2−	4.2 2.2	Mitochondria	One of several possible AM ester hydrolysis products
De-esterified salt	3−	−0.7		Hydrolysis products can have both positive and negative charges

¹Esterified indicates that all carboxylic acids are present in esterified form; part de-esterified indicates that at least one ester grouping was hydrolyzed; de-esterified salt indicates total conversion of all esters to carboxylic acids.

²Z is the maximum possible electric charge for the species.

³Log P is the logarithm of the octanol-water partition coefficient.

⁴Membrane permeability predicted using QSAR models summarized in Table 2.

⁵Sites of accumulation predicted using QSAR models summarized in Table 3.

⁶The hydrolysis products of all listed probes except rhod-2 include several compounds that are lipophilic weak acids, hence could accumulate in lysosomes and mitochondria. Moreover, the hydrolysis products of all probes except calcium green-1 and rhod-2 include lipophilic and moderately amphiphilic compounds that might accumulate in the endoplasmic reticulum; because the presence of such a species cannot be assumed, however, accumulation in endoplasmic reticulum cannot be predicted reliably.

Table 2. Decision logic table based on structured parameters and published QSAR models for predicting membrane permeability and binding to plasma membrane of calcium probes

If	And if	And if	Then
log P > 8			Impermeable; trapped in plasma membrane
CBN > 40			Impermeable; trapped in plasma membrane
AI > 8			Impermeable; trapped in plasma membrane
8 > log P > 0	CBN < 40	AI < 8	Permeable; enters cytosol
log P < 0	CBN < 40		Impermeable; excluded

Table 3. Decision logic table based on numerical structure parameters and published QSAR models for predicting compartmentalization of calcium probes in cell organelles. For models and parameters, see Horobin (2010)

If	And if	And if	And if	Then probe will accumulate in [mechanistic thumbnail]
Z = 0	5 > log P > 0	CBN < 40	AI < 3.5	Cytosol [residual location]
6 > log P > 0	6 > AI > 3.5			Endoplasmic reticulum [fluid membrane partitioning]
8 > log P > 5	Or if 8 > AI > 5			Generic biomembranes [partitioning]
Z > 0	0 > log Pcation > −5	10 > pKa > 6		Lysosomes [weak base ion-trapping]
Zmost ionized < 0	log Pfree acid > 0	pKa = 7 ± 3		Lysosomes [precipitation trapping]
Log P < 0	CBN < 40		Or if CBN > 40 & log P < −10	Lysosomes [uptake by pinocytosis]
Z > 0	5 > log P > 0	pKa > 12		Mitochondria [electrical potential and/or cardiolipin complexation]
Zmost ionized < 0	5 > log Pleast ionized > 0	pKa = 7 ± 3		Mitochondria [weak acid ion-trapping]
Zmost ionized < 0 Or if Z < 0	CBN > 40 40 > CBN > 16	log P > −10 8 > log Panion > 0		Nucleus [histone binding]

Table 4. Examples of compartmentalization following application of the AM ester forms of some commonly used calcium probes

Dye	Organelle in which dye accumulated	Example references
Calcein AM	Endosome/lysosome	Crowe et al. (1995)
	Mitochondrion	Collins et al. (2002), Dedkova and Blatter (2005)
Calcium green-1 AM	Mitochondrion	Babcock et al. (1997), Thomas et al. (2000)
	Nucleus	Babcock et al. (1997), Thomas et al. (2000)
Calcium orange AM	Endoplasmic reticulum	Thomas et al. (2000)
	Mitochondrion	Thomas et al. (2000)
Fluo-3 AM	Endoplasmic reticulum	Thomas et al. (2000)
	Endo-/lysosome	Kuba et al. (1991), Nathanson and Burgstahlen (1992), Thomas et al. (2000)
	Mitochondrion	Thomas et al. (2000)
	Nucleus	Peng et al. (2009), Thomas et al. (2000)
Fura-2 AM	Endoplasmic reticulum	Williams et al. (1985)
	Endosome/lysosome	Di Virgilio et al. (1990), Malgaroli et al. (1987), Roe et al. (1990)
	Mitochondrion	Davis et al. (1987), Roe et al. (1990)
	Nucleus	Roe et al. (1990), Malgaroli et al. (1987), Williams et al. (1985)
Fura red AM	Endoplasmic reticulum	Thomas et al. (2000)
	Mitochondrion	Thomas et al. (2000)
	Nucleus	Broder et al. (2007), Thomas et al. (2000)
Indo-1 AM	Mitochondrion	Lee et al. (1988), Spurgeon et al. (1990)
	Microsome (endoplasmic reticulum?)	Lee et al. (1988), Wahl et al. (1990)
	Nucleus	Lee et al. (1988), Wahl et al. (1990)
Quin-2 AM	Endosome/lysosome	Goligorsky et al. (1986)
Rhod-2 AM	Mitochondrion	Alano et al. (2002), Babcock et al. (1997), Nutt et al. (2002)