Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.
We have developed fluo-4, a new fluorescent dye for quantifying cellular Ca2+ concentrations in the 100 nM to 1 microM range. Fluo-4 is similar in structure and spectral properties to the widely used fluorescent Ca(2+)-indicator dye, fluo-3, but it has certain advantages over fluo-3. Due to its greater absorption near 488 nm, fluo-4 offers substantially brighter fluorescence emission when used with excitation by argon-ion laser or other sources in conjunction with the standard fluorescein filter set. In vitro, fluo-4 exhibited high fluorescence emission, a high rate of cell permeation, and a large dynamic range for reporting [Ca2+] around a Kd(Ca2+) of 345 nM. We have also developed several Ca(2+)-indicators related to fluo-4 having lower affinities for Ca2+ that are useful in cellular studies requiring quantification of higher [Ca2+]. In a variety of physiological studies of live cells, fluo-4 labeled cells more brightly than did fluo-3, when challenged with procedures designed to elevate calcium levels. Fluo-4 is well suited for photometric and imaging applications that make use of confocal laser scanning microscopy, flow cytometry, or spectrofluorometry, or in fluorometric high-throughput microplate screening assays. Because of its higher fluorescence emission intensity, fluo-4 can be used at lower intracellular concentrations, making its use a less invasive practice.
Gee
K R
KR
Molecular Probes, Inc., Eugene, OR 97402, USA. KyleGee@probes.com
Brown
K A
KA
Chen
W N
WN
Bishop-Stewart
J
J
Gray
D
D
Johnson
I
I
D016475:3T3 Cells / Q000378:metabolism; D000814:Aniline Compounds / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D013050:Spectrometry, Fluorescence; D014966:Xanthenes / Q000737:chemistry* / Q000378:metabolism*
D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium