A method for loading equine platelets with the fluorescent calcium indicator Fura-2: ADP induces a rise in the cytosolic free calcium ion concentration
| Autor: | A. W. Poole, R.J. Evans, S. O. Sage, M. F. Heath |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Blood Platelets
Fura-2 chemistry.chemical_element General Medicine Calcium Fluorescence Adenosine Fluorescence spectroscopy Adenosine Diphosphate chemistry.chemical_compound Hydrolysis chemistry Biochemistry Extracellular medicine Biophysics Animals Fluorometry Platelet Chromatography Thin Layer Horses Fluorescent Dyes medicine.drug |
| Zdroj: | Scopus-Elsevier |
| ISSN: | 2042-3306 0425-1644 |
| DOI: | 10.1111/j.2042-3306.1993.tb02900.x |
| Popis: | 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 microM. The technique was validated by demonstrating adequate loading of human platelets with Fura-2, to a concentration of 250-300 microM, 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 microM. Cells loaded as such showed a resting [Ca2+]i of 129 nM and a concentration-dependent rise in [Ca2+]i to a transient maximum of 350 nM when stimulated by the pro-aggregatory agonist adenosine 5'-diphosphate (ADP). |
| Databáze: | OpenAIRE |
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