title: Flow cytometric analysis of calcium mobilization in whole-blood platelets.
abstract: Flow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular free Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. Moreover, in the clinical setting, whole-blood techniques have obvious advantages to avoid artifactual platelet activation and allow the maintenance of near-physiological conditions. This unit describes a fast and sensitive flow cytometric procedure using the Ca2+-sensitive dye fluo-3 AM and the platelet-specific antibody CD41-PE to determine the kinetics of intracellular Ca2+ mobilization in whole-blood platelets with minimal manipulation of the samples. The technique may be applied to reveal fast and transient increases in cytosolic calcium upon platelet stimulation with the agonists ADP and thrombin. This protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal-transduction responses to agonists under near-physiological conditions, and should be broadly applicable to studies of platelet reactivity.
authors.affiliation: Instituto Politécnico de Saüde-Norte, Paredes, Portugal.
authors.forename: Maria-do-Céu
authors.initials: MD
authors.lastname: Monteiro
authors.forename: Maria-José
authors.initials: MJ
authors.lastname: Gonçalves
authors.forename: Filipe
authors.initials: F
authors.lastname: Sansonetty
authors.forename: José-Enrique
authors.initials: JE
authors.lastname: O'Connor
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000032:analysis; D020013:Calcium Signaling; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008722:Methods; D040201:Platelet Membrane Glycoprotein IIb / Q000276:immunology; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D040201:Platelet Membrane Glycoprotein IIb; D014966:Xanthenes; C059715:Fluo-3; D002118:Calcium