1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.
Published January 17, 2012. Keywords: Bioengineering, Fluid Shear Stress, Shear Stress, Shear Force, Endothelium, Endothelial Progenitor Cells, Flow Chamber, Laminar Flow, Flow Circuit, Continuous Flow, Cell Adhesion
JoVE Immunology and Infection
1School of Clinical and Experimental Medicine, University of Birmingham, 2College of Medical and Dental Sciences, University of Birmingham, 3School of Immunity and Infection, University of Birmingham
The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.
Published January 7, 2015. Keywords: Immunology, Endothelial cells, leukocytes, mesenchymal stromal cells, mesenchymal stem cells, co-culture, adhesion, inflammation, recruitment, flow based adhesion assay, Ibidi microslide, neutrophil
JoVE Immunology and Infection
1Genetics and Genomic Sciences Graduate Program, University of Alabama at Birmingham, 2Birmingham Veterans Affairs Medical Center, 3Department of Pathology, University of Alabama at Birmingham, 4Department of Biomedical Engineering, University of Alabama at Birmingham, 5Department of Medicine, University of Alabama at Birmingham
A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.
Published July 2, 2014. Keywords: Immunology, neutrophil adhesion, flow chamber, human umbilical vein endothelial cell (HUVEC), purified ligand
1Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, 2Center for Regenerative Therapeutics, Brigham and Women's Hospital, 3Harvard Medical School, Harvard University, 4Harvard Stem Cell Institute, Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering, Massachusetts Institute of Technology
This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.
Published October 16, 2013. Keywords: Bioengineering, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School
This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.
Published February 11, 2009. Keywords: Immunology, Rolling, Selectins, Endothelial Cells, Parallel-Plate Flow Chamber, Laminar Flow, Shear Stress, Homing, Trafficking, BMEC, bone marrow
1Department of Chemical and Biomolecular Engineering, Russ College of Engineering and Technology, Ohio University, 2Biomedical Engineering Program, Ohio University
Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.
Published September 4, 2013. Keywords: Bioengineering, Cellular Biology, Biomedical Engineering, Molecular Biology, Biophysics, Biochemistry, Chemical Engineering, Chemistry, Cell Adhesion Molecules, Biological Markers, Antigens, Cell Adhesion Molecules, Microscopy, Fluorescence, Cell Physiological Processes, Cell Adhesion, Cell Physiological Phenomena, Colocalization, cell rolling, two-color fluorescence, cell, imaging
1Department of Biomedical Engineering, University of California, Davis
Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.
Published July 21, 2012. Keywords: Biomedical Engineering, Bioengineering, Immunology, Molecular Biology, Genetics, endothelial cell, monocyte arrest, microfluidics, shear stress, cytokine, atherosclerosis, inflammation
JoVE Immunology and Infection
1Department of Physiology and Pharmacology, University of Calgary
This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.
Published August 8, 2012. Keywords: Immunology, Medicine, Physiology, Cellular Biology, HUVEC, ibidi, leukocyte recruitment, neutrophil, flow chamber
1Biomedical Technology, CFD Research Corporation
Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.
Published May 25, 2014. Keywords: Bioengineering, particle, adhesion, shear, microfluidics, vasculature, networks
1Department of Medicine, Weill Cornell Medical College, 2Department of Urology, Weill Cornell Medical College
Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.
Published May 15, 2014. Keywords: Medicine, E-selectin, Metastasis, Microslides, Circulating tumor cells, PSMA, Prostate cancer, rolling velocity, immunostaining, HUVECs, flow chambers
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here's a look at what's coming up in the September 2013 issue of JoVE: The Journal of Visualized Experiments.
Published September 3, 2013. Keywords: This Month in JoVE,
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
Published October 20, 2011. Keywords: Medicine, fluorescence in situ hybridization, FISH, confocal laser scanning microscopy, CLSM, orthodontic appliances, oral biofilm