Calcium Detection Questions
dick at probes.com
Mon Mar 17 09:00:52 EST 1997
Fura-2 is useful only in live cells. Fixation of fura-2 (or all other
calcium indicators) is likely to result in leakage of the low molecular
weight dye. Moreover, the spatial pattern of calcium distribution will
be lost even if it is fixable. Also, if it were fixable, it would
remain calcium sensitive and its fluorescence properties would be
dictated by the calcium concentration in the medium not of the treatment
that proceeded. Above about 1 uM, fura-2 is totally saturated with
Either ionomycin or 4-bromo A-23187 are used with fura-2 in live cells.
A-23187 itself is too fluorescent to use in combination with fura-2.
If you decide to do the experiments in live cells, then I suggest fluo-3
instead of fura-2. Fura-2 is a very good indicator but it is usually
used where one wants to actually measure a calcium concentration and
that is actually not that easy to do. Fluo-3 give nice kinetic results
because of its larger fluorescence change on increasing calcium levels.
Also, it is usually easier for a student to understand changes in
fluorescence emission intensity than shifts in excitation spectra (as in
fura-2). The student must load the cells first (usually about 30
minutes at 37 deg followed by washing) THEN add the ionophore or
whatever else is used to induce the calcium change. The change is
usually rapid (certainly within a few minutes). The cells may remain
useful for an hour or more but may require another washing to remove the
little amount of dye that leaks (this wash step is ESSENTIAL if the
measurement is in a cuvette rather than under a microscope since
indicator that leaks is often into a high calcium-containing medium).
My Handbook is one place to look for information on fluorescence
histochemistry (see PODF files at http://www.probes.com or request a
free copy from Molecular Probes). Particularly good fluorescence
microscopy experiments for students include viability assays (LIVE/DEAD)
and, for instance, detecting live and dead bacteria on cheek scrapings
(see picture in Color Plate 4 of the Handbook). Also, it is
particularly easy to stain mitochondria in live cells with rhodamine 123
and actin in fixed preparations with a variety of fluorescent phalloidin
derivatives (commonly rhodamine phalloidin). Protocols for most of these
and other possible experiments are in Product Information Sheets at the
I obviously DO have commercial connections with Molecular Probes.
Dick Haugland, President
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