Objective To set up a method to determine cytoplasmic free calcium [Ca~(2+)]_(i) in human platelets with flow cytometry.Methods As a calcium indicator,Fluo 3-AM was used to determine the changes,induced by thrombin,of the level of platelet plasma free calcium [Ca~(2+)]_(i) in presence or absence of outside calcium [Ca~(2+)]_(o),in order to elucidate where the platelet plasma free calcium [Ca~(2+)]_(i) mainly come from and it's role in the activity of platelets.Results The geometric mean of fluorescence intensity of the platelet,labeled with Fluo 3-AM,was increased obviously.The concentration of platelet plasma free calcium [Ca~(2+)]_(i) was increased significantly by thrombin in a dose-and [Ca~(2+)]_(o)-dependent manner.Conclusion The increased level of platelet plasma free calcium [Ca~(2+)]_(o),induced by thrombin,mainly comes from [Ca~(2+)]_(o) and partly from the released [Ca~(2+)]_o of intra-platelet probably.

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