Apr 23, 1999

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

Cytometry
M do Céu MonteiroJ E O'Connor

Abstract

Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to ...Continue Reading

  • References13
  • Citations30

Mentioned in this Paper

Monoclonal Antibodies
ITGA2B gene
Flow Cytometry
Adenosine Diphosphate Measurement
Platelet Aggregation Measurement
Calcium
Protoplasm
Aggregation
F2 gene
Intracellular Fluid

About this Paper

Related Feeds

Calcium & Bioenergetics

Bioenergetic processes, including cellular respiration and photosynthesis, concern the transformation of energy by cells. Here is the latest research on the role of calcium in bioenergetics.