Laser scanning cytometry: a novel method for the detection of platelet--endothelial cell adhesion.
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.
Claytor
R B
RB
Department of Surgery, Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. brannon.claytor@umassmed.edu
Li
J M
JM
Furman
M I
MI
Garnette
C S
CS
Rohrer
M J
MJ
Barnard
M R
MR
Krueger
L A
LA
Frelinger
A L
AL
Michelson
A D
AD
D000244:Adenosine Diphosphate / Q000494:pharmacology; D019138:Antibodies, Blocking / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D004338:Drug Combinations; D004727:Endothelium / Q000166:cytology / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D004837:Epinephrine / Q000494:pharmacology; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D010973:Platelet Adhesiveness / Q000187:drug effects / Q000502:physiology*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D004338:Drug Combinations; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000244:Adenosine Diphosphate; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D004837:Epinephrine