Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

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Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow. / Jagau, Hilger; Behrens, Ina Kristin; Steinert, Michael; Bergmann, Simone.

I: Journal of visualized experiments : JoVE, Nr. 152, e60323, 2019.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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Jagau, Hilger ; Behrens, Ina Kristin ; Steinert, Michael ; Bergmann, Simone. / Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow. I: Journal of visualized experiments : JoVE. 2019 ; Nr. 152.

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TY - JOUR

T1 - Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

AU - Jagau, Hilger

AU - Behrens, Ina Kristin

AU - Steinert, Michael

AU - Bergmann, Simone

PY - 2019

Y1 - 2019

N2 - Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells. The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

AB - Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells. The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

U2 - 10.3791/60323

DO - 10.3791/60323

M3 - Article

C2 - 31736484

AN - SCOPUS:85075114856

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 152

M1 - e60323

ER -