Calcium is an important
intracellular messenger ion responsible for the activation and
deactivation of numerous biological events in cells. To study
the role of calcium in these events, one needs to be able to
quantitatively monitor its concentration. The most widely used
method of Ca2+ monitoring is by the use of
fluorescentCa2+ indicators, a technique pioneered by
professor Roger Tsien and colleagues (Tsien, R. in Methods in
Cell Biology, Vol. 30, Taylor, D.L. and Wang, Y-L, Eds.,
Academic Press (1989) pp. 127-156). These indicators probe Ca2+
concentration via their fluorescent spectral changes upon Ca2+
binding. We offer a number of Ca2+ indicators,
including Fura-2, Indo-1, Fluo-3 and Rhod-2.
These products are available in both the membrane-impermeant
salt forms and the membrane-permeant AM ester forms. For each Ca2+
indicator, we have three different salt forms (NH4+,
Na+ and K+) available. All three salt
forms have the same Ca2+ response for a given
indicator. However, you may want to select a salt form that is
compatible with your biological system. The salt forms of the
indicators are water-soluble and can be loaded into cells via
microinjection or scrape loading. The AM esters of the
indicators are membrane-permeant and thus can be loaded into
cells by simple incubation of the cell or tissue preparation in
a buffer containing the AM ester. We also supply Pluronic
F-127(59000), a mild nonionic detergent that can facilitate the
loading of the AM esters. Pluronic F-127 is offered FREE with
each order of any AM esters. The AM esters themselves do not
bind Ca2+ . However, once they have entered the
cells, they are readily hydrolyzed by intracellular esterases
into the parent Ca2+ indicators, thus becoming responsive to Ca2+
.
The primary differences among
Fura-2, Indo-1, Fluo-3 and Rhod-2
are in their Ca2+ dissociation constants (Kd)
or Ca2+ response range, excitation/emission
wavelengths, spectral shift, and relative fluorescent quantum
yields. Therefore, you should select a Ca2+ indicator
that best suits your need in consideration of your biological
system, instrument settings and any other fluorescent probes
that you may use at the same time. The Kd values can give you an
estimate of the detectable Ca2+ concentration range,
usually 0.1Kd to 10Kd. However, one should
be cautious in using these in vitro determined Kd
values as the Kd values in cells could differ considerably due
to differences in ionic strength, pH, viscosity and Ca2+
buffering by intracellular lipids and proteins (Petr, M.J. and
Wurster, R.D. Cell Calcium, 21, 233(1997)). |
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