Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets

Maria‐do‐Céu Monteiro1, Maria‐José Gonçalves1, Filipe Sansonetty2, José‐Enrique O'Connor3

1 Instituto Politécnico de Saüde‐Norte, Paredes, Portugal, 2 Escola de Ciências de Saúde, Universidade do Minho, Braga, Portugal, 3 Centro de Citometría y Citómica, Universidad de Valencia, Valencia, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.20
DOI:  10.1002/0471142956.cy0920s24
Online Posting Date:  May, 2003
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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.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Rested donor free from antiplatelet treatment
  • 135 mM tribasic sodium citrate, pH 7.4
  • Modified Tyrode's buffer (see recipe)
  • 1 mM fluo‐3 AM (see recipe)
  • CD41‐PE monoclonal antibody (Immunotech)
  • 5 mg/ml glycyl‐L‐prolyl‐L‐arginyl‐L‐proline (GPRP; see recipe); for thrombin activation studies
  • Agonist (e.g., human α‐thrombin, ADP; see recipe)
  • 19‐G needle
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with 488‐nm excitation and band‐pass filters centered at 525 (green) and 575 nm (orange)
NOTE: Use new or carefully washed polypropylene tubes in all steps to avoid platelet activation on glass or polystyrene surfaces.NOTE: The reader is encouraged to read unit 6.10 for more detail on platelet handling and unit 9.8 for data analysis.
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Literature Cited

Literature Cited
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   Bennett, J.S. and Kolodziej, M.A. 1992. Disorders of platelet function. Dis. Mon. 38:577‐631.
   Davies, T.A., Drotts, D., Weil, G.J., and Simons, E.R. 1988. Flow cytometric measurements of cytoplasmic calcium changes in human platelets. Cytometry 9:138‐142.
   Davies, T.A., Drotts, D.L., Weil, G.J., and Simons, E.R. 1989. Cytoplasmatic Ca2+ is necessary for thrombin‐induced platelet activation. J. Biol. Chem. 264:19600‐19606.
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   Monteiro, M.C., Sansonetty, F., Gonçalves, M.J., and O'Connor, J.E. 1999. Flow cytometric kinetic assay of calcium mobilization in whole‐blood platelets using fluo‐3 and CD41. Cytometry 35:302‐310.
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