Biology Reference
In-Depth Information
the Ca
2
þ
-sensitive form of the indicator when incubated with micromolar concen-
trations of the AM ester in the extracellular medium. Several factors influence the
e
ciency and quality of indicator loading via the AM ester, and will be discussed
subsequently.
Y
A. Limited Aqueous Solubility of AM Esters
AM esters of the common Ca
2
þ
indicators have molecular weights in excess of
1000. Being large uncharged organic molecules, these esters have very low solubili-
ty in aqueous media. For example, at 25
C, the solubility of the AM ester of Fura-
2 in pure water is only 0.11
m
M(
Kao et al., 1990
). In biological media, in which the
ionic strength is typically
0.15 M, the solubility of Fura-2 AM would be even
lower. Addition of AM ester in excess of the solubility limit simply would result in
precipitation of solid AM ester, which is e
ectively unavailable for loading cells. In
addition, fine particles of solid AM ester often adhere well to the outer surfaces of
cells or to the extracellular matrix and can contribute large Ca
2
þ
-insensitive
fluorescence signals to the measurement.
6
A convenient solution to the solubility
problem is the use of Pluronic F-127, a mild nonionic surfactant,
7
as a dispersing
agent for AM esters. Typically, aliquots of Pluronic and AM ester stock solutions
in dimethylsulfoxide (DMSO) are mixed intimately before dispersal into an aque-
ous medium.
8
The Pluronic is presumed to sequester the AM ester in micellar form,
thus preventing precipitation, and the micelles serve as a steady source to replenish
AM esters taken up by cells. The net result is significantly improved loading of
indicators into cells. Details of the loading procedure are described in
Section III.D
below.
V
B. Dye Compartmentalization: Loading of Indicator into Subcellular Compartments
Other than the Cytosol
1. Minimizing Compartmentalization
In typical experiments, one usually wishes to monitor changes in the concen-
tration of Ca
2
þ
in the cytosol; therefore, ideally, one would like the indicator to
be loaded exclusively into the cytosol. This ideal situation is almost never
realized for two reasons. First, because the AM ester form of the indicator is
membrane-permeant,
it can enter not only the cytosol but all subcellular
6
This is a problemwith AM ester that are fluorescent (e.g., Fura-2 AMand Indo-1 AM) but not with
nonfluorescent AM esters (e.g., AM ester of the Fluo series).
7
Pluronic F-127 is manufactured by BASF Wyandotte Corporation, Wyandotte, Wisconsin. Being
a surfactant used on the industrial scale, it is inexpensive.
8
With slight warming, Pluronic F-127 can be dissolved in DMSO at almost 25% (w/v). Such a highly
concentrated stock solution is inconvenient to use, however: as the solution absorbs moisture from the
air, the Pluronic will precipitate, and the resulting particulate suspension is di
Y
cult to pipette. A 15-20%
solution in DMSO is a convenient formulation.
