Laser scanning cytometry: a novel method for the detection of platelet--endothelial cell adhesion

Cytometry. 2001 Apr 1;43(4):308-13. doi: 10.1002/1097-0320(20010401)43:4<308::aid-cyto1063>3.0.co;2-9.

Abstract

Background: Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets.

Methods: Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals.

Results: LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion.

Conclusions: LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.

MeSH terms

  • Adenosine Diphosphate / pharmacology
  • Antibodies, Blocking / pharmacology
  • Antibodies, Monoclonal / pharmacology
  • Blood Platelets / drug effects
  • Blood Platelets / physiology
  • Blood Platelets / ultrastructure
  • Cells, Cultured
  • Drug Combinations
  • Endothelium / cytology
  • Endothelium / physiology
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / physiology*
  • Epinephrine / pharmacology
  • Humans
  • Image Cytometry / methods*
  • Microscopy, Confocal / methods*
  • Platelet Adhesiveness / drug effects
  • Platelet Adhesiveness / physiology*
  • Platelet Glycoprotein GPIIb-IIIa Complex / immunology
  • Tetradecanoylphorbol Acetate / pharmacology
  • Thrombin / pharmacology

Substances

  • Antibodies, Blocking
  • Antibodies, Monoclonal
  • Drug Combinations
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Adenosine Diphosphate
  • Thrombin
  • Tetradecanoylphorbol Acetate
  • Epinephrine