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 Phone: (517) 349-7200 FAX: (517) 349-5967 e-mail: [log in to unmask]