Unit

UNIT 9.20 Flow Cytometric Analysis of Calcium Mobilization in Whole-Blood Platelets

  1. Maria-do-Céu Monteiro1,
  2. Maria-José Gonçalves1,
  3. Filipe Sansonetty2,
  4. José-Enrique O'Connor3

Published Online: 1 MAY 2003

DOI: 10.1002/0471142956.cy0920s24

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Monteiro, M.-d.-C., Gonçalves, M.-J., Sansonetty, F. and O'Connor, J.-E. 2003. Flow Cytometric Analysis of Calcium Mobilization in Whole-Blood Platelets. Current Protocols in Cytometry. 24:9.20:9.20.1–9.20.8.

Author Information

  1. 1

    Instituto Politécnico de Saüde-Norte, Paredes, Portugal

  2. 2

    Escola de Ciências de Saúde, Universidade do Minho, IPATIMUP, Braga, Portugal

  3. 3

    Centro de Citometría y Citómica, Universidad de Valencia, Valencia, Spain

Publication History

  1. Published Online: 1 MAY 2003
  2. Published Print: APR 2003

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.