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Indo-1 and Ca++ measurments. What is the general feeling for the
"best"
fluorescent indicator in PBL's or cultured T-cell lines? Is Indo-1
the
current indicator of choice? Is it the best probe to use to look for
T cell
activation/response to stimulators? I have read about many of the
new
probes being developed, especially those excited in the visible
spectrum,
and am curious if they would be better and easier to use than the
Indo-1.
Any and all comments would be appreciated.
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The brief answer for indicator of calcium flux is probably yes, but
it depends on what you wish to find out and the kind of instrument at
your disposal.
Taking the instrument issue first, you can only use indo-1 if you
have an instrument that has an excitation source in the uv (and, of
course, the appropriate filters for fluorescence); uv excitation
sources could be either an argon or krypton water-cooled laser (with
uv optics), an air-cooled helium cadmium laser, or a mercury arc
lamp. A bench top analyzer with only an air-cooled argon laser will
limit your choices to fluo-3, calcium green, orange, or crimson.
Fluo-3 is the only probe with published work in flow cytometry.
If the purpose is to demonstrate that there is a difference in the
rate of calcium flux following stimulation of the cells then many of
the probes will probably work. But if you want to get a more precise
estimate of the calcium concentration within cells, then a
ratiometric method is by far the best choice and indo-1 is probably
the preferred probe to use in flow cytometry as described below.
Fluo-3 can be used ratiometrically if another probe is available that
does not change with experimental manipulation. There are several
assumptions that must be satisfied. This seemed to work with snarf-1
in the paper by John Cambier and others a couple of years ago.
(Indo-1 is fairly susceptible to photobleaching so imaging
applications over a time period have some problems. Roger Tsien is
working on more bleach-resistant probes.)
Since you have instruments that have argon lasers capable of uv
emission, then indo-1 is probably the best choice for estimating
calcium levels using flow cytometry the following reasons:
1. Indo-1 is a ratiometric probe, thus the inference of
calcium on a cell by cell basis is independent of the absolute
brightness of the fluorescence. So things like dye loading, cell
size, etc. are not variables that will confound the measurement
2. There is a lot a experience with indo-1 making it probably
the "gold standard" (in so far as one exists) for calcium measurement
in flow.
3. The loading conditions are fairly well defined. This will
vary with cell type, but the variations are small and fairly easily
performed.
4. With a multi-laser instrument it's not difficult to use
indo-1 in conjunction with several other probes. For example, most
antibody labels will be excited by 488. Much more meaningful
measurements can be done if different phenotypes can be defined
simultaneously.
The calibration of the ratio for a particular instrument is at best
an estimate for two reasons: the actual calibration is not
straightforward (this may change), and the Kd of indo-1 for Ca2+ is a
function of the ionic and pH environments. If these vary within
cellular compartments (this cannot be known) then the apparent
calcium measurements will be in error if the calibration assumes a
different value.
Dave Coder
Dept. of Immunology
Univ. Washington
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