Human Neutrophil Flow Chamber Adhesion Assay

The vascular endothelium interacts with all types of blood cells and is a key modulator of local and systemic inflammatory processes, for example, in the adhesion of blood leukocytes to endothelial cells (EC) and the following extravasation into the injured tissue. The endothelium is constantly exposed to mechanical forces caused by blood flow, and the resulting shear stress is essential for the maintenance of endothelial function. Changes in local hemodynamics are sensed by EC, leading to acute or persistent changes. Therefore, in vitro assessment of EC functionality should include shear stress as an essential parameter. Parallel-plate flow chambers with adjustable shear stress can be used to study EC properties. However, commercially available systems are not suitable for radiation experiments, especially with charged particles, which are increasingly used in radiotherapy of tumors. Therefore, research on charged-particle-induced vascular side effects is needed. In addition, α-particle emitters (e.g., radon) are used to treat inflammatory diseases at low doses. In the present study, we established a flow chamber system, applicable for the investigation of radiation induced changes in the adhesion of lymphocytes to EC as readout for the onset of an inflammatory reaction or the modification of a pre-existing inflammatory state. In this system, primary human EC are cultured under physiological laminar shear stress, subjected to a proinflammatory treatment and/or irradiation with X-rays or charged particles, followed by a coincubation with primary human lymphocytes (peripheral blood lymphocytes (PBL)). Analysis is performed by semiautomated quantification of fluorescent staining in microscopic pictures. First results obtained after irradiation with X-rays or helium ions indicate decreased adhesion of PBL to EC under laminar conditions for both radiation qualities, whereas adhesion of PBL under static conditions is not clearly affected by irradiation. Under static conditions, no radiation-induced changes in surface expression of adhesion molecules and activation of nuclear factor kappa B (NF-κB) signaling were observed after single cell-based high-throughput analysis. In subsequent studies, these investigations will be extended to laminar conditions.

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“…Using this approach, we applied a shear stress of 0.75 dyn/cm 2 , which is in the range of physiological blood flow in small vessels (34). …”

Section: Methodsmentioning

confidence: 99%

Measuring Leukocyte Adhesion to (Primary) Endothelial Cells after Photon and Charged Particle Exposure with a Dedicated Laminar Flow Chamber

et al. 2017

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“…In these settings, PBMCs were then incubated with HUVECs that were only activated with TNFα, but no additional MnTBAP. For the dynamic adhesion assay of neutrophils, neutrophils were isolated with heavy and light Histopaque® (Histopaque-1077 and Histopaque-1119; Sigma-Aldrich) as previously described [13]. The GlykoTech flow chamber was assembled with the dish at the bottom of the resulting parallel flow chamber.…”

Section: Methodsmentioning

confidence: 99%

MnTBAP increases BMPR-II expression in endothelial cells and attenuates vascular inflammation

Zhou

Einert

Schmitt

et al. 2016

Vascular Pharmacology

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Aims The endothelium plays an important role during vascular inflammation. Previous data have demonstrated a high expression level of manganese-superoxide dismutase (MnSOD) in endothelial cells and suggested an important role of MnSOD in several cardiovascular diseases. Manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) has been shown to mimic some of the effects of MnSOD and prevented the development of diabetes and obesity. However, its effect on vascular inflammation and the underlying mechanism is still unknown. Methods and results Leukocyte adhesion was evaluated in-vivo and in-vitro using dynamic flow chamber and intravital microscopy in mice. Expression of adhesion molecules induced by TNFα and adhesion of leukocytes to the vessel wall were inhibited by MnTBAP. The anti-inflammatory effect of MnTBAP was partly mediated by up-regulation of the BMPR-II and Smad dependent pathway. Additionally, MnTBAP decelerated the turn-over of endogenous BMPR-II. Conclusion Our data demonstrate that MnTBAP activates Smad signaling, preserves the turn-over of BMPR-II and elicits anti-inflammatory effects in endothelial cells, partly mediated by BMPR-II. This finding suggests a potential therapeutic impact of MnTBAP in the treatment of vascular inflammation.

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“…22 In this experiment, V was estimated to be 7.8 mm/s. The total volume of naturally occurring air bubbles during 1 week of culture was estimated to be 20.16 mm 3 .…”

Section: Optimization Of the Bubble Trapmentioning

confidence: 79%

An Easy-to-Use Polystyrene Microchip-based Cell Culture System

Tazawa¹,

Sunaoshi

Tokeshi

et al. 2016

ANAL. SCI.

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In this study, we developed an integrated, low-cost microfluidic cell culture system that is easy to use. This system consists of a disposable polystyrene microchip, a polytetrafluoroethylene valve, an air bubble trap, and an indium tin oxide temperature controller. Valve pressure resistance was validated with a manometer to be 3 MPa. The trap protected against bubble contamination. The temperature controller enabled the culture of Macaca mulatta RF/6A 135 vascular endothelial cells, which are difficult to culture in glass microchips, without a CO2 incubator. We determined the optimal coating conditions for these cells and were able to achieve stable, confluent culture within 1 week. This practical system is suitable for low-cost screening and has potential applications as circulatory cell culture systems and research platforms in cell biology.

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Human Neutrophil Flow Chamber Adhesion Assay

Cited by 13 publications

References 22 publications

Measuring Leukocyte Adhesion to (Primary) Endothelial Cells after Photon and Charged Particle Exposure with a Dedicated Laminar Flow Chamber

Measuring Leukocyte Adhesion to (Primary) Endothelial Cells after Photon and Charged Particle Exposure with a Dedicated Laminar Flow Chamber

MnTBAP increases BMPR-II expression in endothelial cells and attenuates vascular inflammation

An Easy-to-Use Polystyrene Microchip-based Cell Culture System

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