On Mon, 29 Jan 1996 09:27:28 -0500 you wrote:
 
>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
>
>*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*
>  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
>*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*-*
>
>
Your Calcium Flow chart is quite interesting.  I would suggest a few
modifications.  We have found virtually no instance of Indo-1AM
INsensitive product examining multiple cell lines.  I think this
problem is much more prevalent in Fura-2 loaded cells. I agree that
it should be the dye of choice if you have UV available and two
detectors to ratio.
 
Also, I think Fura-2 ought to be mentioned.  Although it's not
appropriate for most confocals that utilize a laser because it
requires 340 and 380 excitation, perhaps the basics about its
usefulness should be documented.  Many people considering doing
calcium experiments have heard of Fura-2 and not some of the other
probes.
 
If one can normalize the Fluo-3 data, useful comparisons between
cells and their responses are more easily accomplished than comparing
raw data.
 
Calcium Green should be included in the visible, single emission
probe list with Fluo-3 as another alternative.  Although similar data
is obtained, it is documented to be less bleachable and several times
brighter at higher calcium levels than Fluo-3.
 
Peggy Wade
Applications Development
 
 
Meridian Instruments, Inc.
Okemos, MI 48864
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