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A method for loading equine platelets with the fluorescent calcium indicator Fura‐2: ADP induces a rise in the cytosolic free calcium ion concentration
Author(s) -
POOLE A. W.,
HEATH M. F.,
SAGE S. O.,
EVANS R. J.
Publication year - 1993
Publication title -
equine veterinary journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.82
H-Index - 87
eISSN - 2042-3306
pISSN - 0425-1644
DOI - 10.1111/j.2042-3306.1993.tb02900.x
Subject(s) - fura 2 , chemistry , platelet , fluorescence , calcium , adenosine diphosphate , extracellular , cytosol , chromatography , biophysics , fluorescence spectroscopy , biochemistry , platelet aggregation , medicine , enzyme , biology , physics , organic chemistry , quantum mechanics
Summary Equine platelets in platelet‐rich plasma were incubated with the fluorescent indicator dye, Fura‐2‐AM (Fura‐2‐acetoxymethyl ester) and the degree of loading of the cells with the dye and the extent of hydrolysis of the ester was assessed by quantitative fluorimetry and by thin‐layer chromatography respectively. Under these conditions the cells loaded poorly with Fura‐2 to a concentration of 4 μM. The technique was validated by demonstrating adequate loading of human platelets with Fura‐2, to a concentration of 250–300 μM, using the same method. The removal of plasma from the extracellular medium was important for successful loading of the cells with the dye since washed cells resuspended in a physiological salt solution loaded adequately with Fura‐2 to a concentration of 190 μM. Cells loaded as such showed a resting [Ca 2+ ] i of 129 nM and a concentration‐dependent rise in [Ca 2+ ] i to a transient maximum of 350 nM when stimulated by the pro‐aggregatory agonist adenosine 5′‐diphosphate (ADP).