I would like to generate a concensus about the best ways to measure
calcium in the confocal. I would like to propose a flow sheet of choices
and then let everyone rip it to shreds (this is what friends are for...).
 
Here is my proposed flow chart for dye selection.....
 
1. INDO-1 is the first line choice of calcium-sensitive dye because of
dual-emission ratios. INDO-1/AM is a crummy esterase substrate, but if
you get enough calcium-sensitive fluorescence in your cells it will work.
Major hassle is calcium-INsensitive fluorescence from the remaining
INDO-1/AM (sometimes reduced by post incubation without dye). INDO-1 also
requires a UV laser. When loading is poor or you have no UV laser, move
on to other choices. Only exception is if you REALLY need quantitative
results, because as you leave INDO-1 you move into more qualitative
methods: in this case invest time in optimizing dye loading.
 
2. FLUO-3 and FURA-Red (or some such combination) is the next best choice.
This method of dual dye loading can be semi-quantitative over
short time intervals. Each days loading is different, so must normalize
in some way to compare results day-to-day (eg. to ratio in resting cells
before "stimulation"). Must prove that dye loss/photobleaching is
negligible (or similar between probes) over the time course of study. A
major advantage of these dyes is that there is little or no fluorescence
from AM forms, and the higher exciatation and emission spectra keep
autofluorescence to a minimum. In the confocal, differences in
compartmentalization between dyes may not destroy results IF dye does not
shuttle between compartments during stimulation (i.e. ratios at any
cellular site should be valid, but don't compare results in the nucleus
versus results in cytoplasm unless you can prove [dye] is about the same in
each compartment).
 
3. FLUO-3 alone is the simplest method for identifying sites of calcium
mobilization. Use the method when this is your only goal: it is only
qualitative because there is no ability to do ratioing in any form. Good
for recognition of calcium mobilization (since fluorescence increases it
can not be ascribed to common artifacts of dye loss/bleach). Fluo-3 is a
good choice because the 488 line (and common FITC filters) make it
accessible in most confocals.
 
So that is my suggested scenario, ready to be "adjusted" by you all. and
now a few questions for the calcium jocks....
 
Are there other (new/old) dyes which should be put into this flow chart
because they are better alternatives than the ones I suggested?
 
Are my decision points for dye switches correct? Are there other
important components to the decision?
 
Are there alternative techniques to the dual dye loading for
semi-quantitative work (which most of us are interested in)? For instance
can you use fluorescence lifetime measurements in some way to quantify
calcium, similar to the pH dyes?
 
Thanks all. Chip
 
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  Chip Montrose
   Johns Hopkins University     tel: 410-955-9681
    Ross 930                      FAX: (410) 955-9677
     720 Rutland Avenue            email: [log in to unmask]
      Baltimore, MD 21205
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