A method for loading equine platelets with the fluorescent calcium indicator Fura-2: ADP induces a rise in the cytosolic free calcium ion concentration.
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).
Poole
A W
AW
University of Cambridge, Department of Clinical Veterinary Medicine, UK.
Heath
M F
MF
Sage
S O
SO
Evans
R J
RJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D002855:Chromatography, Thin Layer / Q000662:veterinary; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000662:veterinary; D016257:Fura-2 / Q000031:analogs & derivatives*; D006736:Horses / Q000097:blood*
D005456:Fluorescent Dyes; C049925:fura-2-am; D000244:Adenosine Diphosphate; D002118:Calcium; D016257:Fura-2