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Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+.
- Source :
-
The Biochemical journal [Biochem J] 1990 Jul 15; Vol. 269 (2), pp. 513-9. - Publication Year :
- 1990
-
Abstract
- A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.
- Subjects :
- Benzofurans
Blood
Blood Platelets ultrastructure
Buffers
Digitonin pharmacology
Egtazic Acid pharmacology
Fura-2
Humans
N-Formylmethionine Leucyl-Phenylalanine pharmacology
Neutrophils ultrastructure
Probenecid pharmacology
Spectrometry, Fluorescence
Thrombin pharmacology
Aniline Compounds
Blood Platelets analysis
Calcium blood
Cytosol analysis
Fluorescent Dyes
Neutrophils analysis
Xanthenes
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 269
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 2117443
- Full Text :
- https://doi.org/10.1042/bj2690513