Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions.
This article intends to close the gap between the abundance of regular articles focusing on adhesive mechanisms of cells in a flow field and purely technical reports confined to the description of newly developed algorithms, not yet ready to be used by users without programming skills. A simple and robust method is presented for analysing raw videomicroscopic data of flow-based adhesion assays using the freely available public domain software ImageJ. We describe in detail the image processing routines used to rapidly and reliably evaluate the number of adherent and translocating platelets in videomicroscopic recordings. The depicted procedures were exemplified by analysing platelet interaction with immobilized von Willebrand factor and fibrinogen in flowing blood under physiological wall shear rates. Neutralizing GPIbalpha function reduced shear-dependent platelet translocation on von Willebrand factor and abolished firm platelet adhesion. Abciximab, Tirofiban and Eptifibatide completely inhibited GPIIb/IIIa-dependent stable platelet deposition on fibrinogen. The presented method to analyse videomicroscopic recordings from flow-based adhesion assays offers the advantage of providing a simple and reliable way to quantify flow-based adhesion assays, which is completely based on ImageJ and can easily be applied to study adhesion mechanisms of cells in non-fluorescent modes without the need to deviate from the presented protocol.
Meyer dos Santos
Sascha
S
Department of Clinical Pharmacology, Klinikum, J.W.Goethe-Universität Frankfurt/Main, Frankfurt am Main, Germany. santos@med.uni-frankfurt.de
Klinkhardt
Ute
U
Schneppenheim
Reinhard
R
Harder
Sebastian
S
D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D039421:Integrin alpha2 / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods*; D010973:Platelet Adhesiveness / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex; D012984:Software; D014841:von Willebrand Factor / Q000378:metabolism
C056564:ITGA2B protein, human; D039421:Integrin alpha2; D039661:Integrin beta3; D008562:Membrane Glycoproteins; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; C094990:adhesion receptor; D014841:von Willebrand Factor; D005340:Fibrinogen