title: 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+.
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.
authors.affiliation: Smith, Kline & French Research Ltd., Welwyn, Herts, U.K.
authors.forename: J E
authors.initials: JE
authors.lastname: Merritt
authors.forename: S A
authors.initials: SA
authors.lastname: McCarthy
authors.forename: M P
authors.initials: MP
authors.lastname: Davies
authors.forename: K E
authors.initials: KE
authors.lastname: Moores
mesh_terms: D000814:Aniline Compounds; D001572:Benzofurans; D001769:Blood; D001792:Blood Platelets / Q000032:analysis* / Q000648:ultrastructure; D002021:Buffers; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000032:analysis*; D004072:Digitonin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000032:analysis* / Q000648:ultrastructure; D011339:Probenecid / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D001572:Benzofurans; D002021:Buffers; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004072:Digitonin; D011339:Probenecid; D002118:Calcium; D016257:Fura-2