Name: pneumococcus infection of primary human endothelial cells in constant flow
Uploaded: 2019-10-31T13:30:00
Description: Watch this Scientific Journal Video about Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow at JoVE.com

The project was funded by the DFG (BE 4570/4-1) to S.B.

The microfluidic cell cultivation was performed with commercial primary human umbilical vein endothelial cells (HUVEC). The company isolated the cells with informed consent of the donor. This study was approved by the Ethics Committee of Doctors Chamber of the Federal State Baden-Wuerttemberg with the reference number 219-04.
NOTE: See Table of Materials for protocol supplies.
1. Precultivation of Primary Endothelial Cells
<ol>
	<...

<ol>
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The simulation of bacterial interaction with mechanosensitive host proteins such as VWF requires a perfusable cell culture system that enables the generation of a defined, unidirectional, and continuous flow of liquids, thereby generating reliable shear stress. Several microfluidic pump systems have already been described. A comprehensive review from Bergmann et al. summarizes the key aspects of different two- and three-dimensional cell culture models17.
The microfluidi...

The pathogenesis of pneumococcus infections is characterized by a multifaceted interaction with a diversity of extracellular matrix compounds and components of the human hemostasis, such as plasminogen and VWF1,2,3,4,5,6,7,8. The multidomain glycoprotein VWF serves as key regulator of a balanced hemostasis by mediating thrombocyte recruitment and fibrin incorporation at the site of vascular thrombus formation9. The importance of functional, active VWF for bleeding control and wound healing is demonstrated by von Willebrand&#8217;s disease, a common inherited bleeding disorder10.
Globular VWF circulates in the human blood system at a concentration of up to 14.0 &#181;g/mL11,10. In response to vascular injury, the local release of VWF by endothelial Weibel Palade Bodies (WBP) is markedly increased11,12. Previous studies show that pneumococcus adherence to human endothelial cells and its production of the pore-forming toxin pneumolysin significantly stimulates luminal VWF secretion13. The hydrodynamic forces of the blood flow induce a structural opening of the mechanoresponsive VWF domains. At flow rates of 10 dyn/cm2 the VWF multimerizes to long protein strings of up to several hundred micrometers in length that remain attached to the subendothelium10,12.
To understand the function of multimerized VWF strings generated under shear stress in the interaction of pneumococcus with the endothelial surface, a microfluidic-based cell culture infection approach was established. A microfluidic device with a software-controlled air-pressure pump system was used. This enabled a continuous, unidirectional recirculation of cell culture medium with a defined flow rate. Thereby, the system applied a defined shear stress on the surface of endothelial cells, which remained attached inside specialized channel slides. This approach enabled the simulation of the shear force within the blood stream of the human vascular system, in which VWF strings are generated on differentiated endothelial cells under defined constant flow conditions. For this purpose, the endothelial cells were cultivated in specific channel slides (see Table of Materials), which were adapted for microscopic analyses during flow. The microfluidic pump system provided the highly defined and controlled shear stress situation required for the formation of extended VWF strings on the confluent endothelial cell layer. After the stimulation of VWF-secretion of confluently grown human umbilical vein endothelial cells (HUVEC) by histamine supplementation, the string formation was induced by applying a shear stress (&#1295;) of 10 dyn/cm2. The shear stress is defined as the force acting on the cell layer. It is calculated approximately according to Cornish et. al.14 with equation 1: 
<img alt="Equation 1" src="/files/ftp_upload/60323/60323equ01.jpg" />
Where &#1295; = shear stress in dyn/cm2, &#951; = viscosity in (dyn&#8729;s)/cm2, h =half of channel height, w = half of channel width, and &#934; = flowrate in mL/min.
The result of equation 1 depends on the different heights and widths of the different slides used (see Table of Materials). In this study a Luer channel slide of 0.4 &#181;m resulting in a chamber slide factor of 131.6 was used (see formula 2). 
<img alt="Equation 2" src="/files/ftp_upload/60323/60323equ02.jpg" />
Viscosity of the medium at 37 &#176;C is 0.0072 dyn&#8729;s/cm&#178; and a shear stress of 10 dyn/cm&#178; was used. This resulted in a flow rate of 10.5 mL/min (see formula 3). 
<img alt="Equation 3" src="/files/ftp_upload/60323/60323equ03.jpg" />
Here, the adaptation and advancement of a microfluidic cell culturing procedure using a unidirectional laminar flow system for the investigation and visualization of bacterial infection mechanisms in the host vasculature is described in detail. The generation of VWF strings on endothelial layers can also be stimulated by using other pump systems that are able to apply a continuous and steady shear stress15.
After cultivation of primary endothelial cells to confluence in flow and stimulation of VWF string formation, pneumococci expressing red fluorescence protein (RFP)16 were added to the endothelial cell layer under constant microscopic control. The attachment of bacteria to VWF strings on the surface of endothelial cells was microscopically visualized and monitored for up to three hours in real time by using VWF-specific fluorescent-labelled antibodies. With this approach, the role of VWF as an adhesion cofactor promoting bacterial attachment to the vascular endothelium was determined8.
In addition to the microscopic visualization of protein secretion and conformational changes, this method could be used to monitor single steps of bacterial infection processes in real time and to quantify the amount of attached bacteria at different time points of infection. The specific software-controlled pump system also provides the possibility to culture the endothelial cells in defined constant flow conditions for up to several days and enables a defined pulsed medium flow incubation. Moreover, this method can be applied using different cell types. Adapting the staining protocol also enables the detection and visualization of bacteria internalized into eukaryotic cells.
This manuscript describes this advanced experimental protocol that can be used as a defined, reliable, and reproducible approach for an efficient and versatile characterization of pathophysiological processes.

<table>
	<tbody>
		<tr>
			<td>1 mL Luer-syringe</td>
			<td>Fisher Scientific</td>
			<td>10303002</td>
			<td>with 1 mL volume for gelatin injection using the luer-connection of the slides</td>
		</tr>
		<tr>
			<td>2 mL Luer-syringe</td>
			<td>Sarstedt</td>
			<td>9077136</td>
			<td>For pieptting/injecting fluids into the luer connections of the channel chamber slides</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>eBioscience now thermo fisher</td>
			<td>00-4555-56</td>
			<td>protease mix used for gentle detachment of endothelial cells</td>
		</tr>
		<tr>
			<td>AlexaFluor350-conjugated Phalloidin</td>
			<td>Abcam</td>
			<td>ab176751</td>
			<td>no concentration available from the manufacturer, stock solution is sufficient for 300 tets, company recommends to use 100 &#181;l of a 1:1000 dilution, blue fluorescence (DAPI-filter settings)</td>
		</tr>
		<tr>
			<td>AlexaFluor488-conjugated goat-derived anti-mouse antibody</td>
			<td>Thermo Fisher Sientific</td>
			<td>A11001</td>
			<td>stock concentration: 2 mg/mL for immunostaining of human VWF in microfluidic slide after PFA fixation, green fluorescence</td>
		</tr>
		<tr>
			<td>AlexaFluor568-conjugated goat-derived anti-rabbit-antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11011</td>
			<td>stock concentration: 2 mg/mL for immunostaining of pneumococci in microfluidic slide after pFA fixation, red fluorescence</td>
		</tr>
		<tr>
			<td>Bacto Todd-Hewitt-Broth</td>
			<td>Becton Dickinson GmbH</td>
			<td>BD 249210</td>
			<td>complex bacterial culture medium</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A2153-25G</td>
			<td>solubilized, for preparation of blocking buffer</td>
		</tr>
		<tr>
			<td>Cell culture flasks with filter</td>
			<td>TPP</td>
			<td>90026</td>
			<td>subcultivation of HUVEC in non-coated cell culture flasks of 25 cm2 surface</td>
		</tr>
		<tr>
			<td>Centrifuge Allegra X-12R</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>392304</td>
			<td>spinning down of bacteria (volumes of &#62; 2mL)</td>
		</tr>
		<tr>
			<td>Centrifuge Allegra X-30</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>B06314</td>
			<td>spinning down of eukaryotic cells</td>
		</tr>
		<tr>
			<td>Centrifuge Z 216 MK</td>
			<td>Hermle</td>
			<td>305.00 V05 - Z 216 M</td>
			<td>spinning down of bacteria (volumes of less than 2 mL)</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>3886.2</td>
			<td>used in a concentration of 0.2 /mL for cultivation of pneumococci transformed with genetic construct carrying red fluorescent protein and chloramphenicol resistance cassette</td>
		</tr>
		<tr>
			<td>Clamp for perfusion tubing</td>
			<td>ibidi</td>
			<td>10821</td>
			<td>for holding the liquid in the tube bevor connecting the slide to the pump system</td>
		</tr>
		<tr>
			<td>CO2-Incubator</td>
			<td>Fisher Scientific</td>
			<td>MIDI 40</td>
			<td>incubator size is perfectly adapted to teh size of the fluidic unit with connected channel slide and was used for flow cultivation at 37&#176;C and 5% CO2</td>
		</tr>
		<tr>
			<td>CO2-Incubator</td>
			<td>Sanyo</td>
			<td>MCO-18 AIC</td>
			<td>for incubation of bacteria and cells in a defined atmosphere at 5% CO2 and 37&#176;C</td>
		</tr>
		<tr>
			<td>Colombia blood agar plates</td>
			<td>Becton Dickinson GmbH</td>
			<td>PA-254005.06</td>
			<td>agar-based complex culture medium for S. pneumoniae supplemented with 7% sheep blood</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Latitude 3440</td>
			<td>Comuter with pressure pump software</td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Microscope (CLSM)</td>
			<td>Leica</td>
			<td>DMi8</td>
			<td>An inverse microscope with a stage covered by a heatable chamber and with a fluorescence unit equipped with fluorescence filter, Xenon-light source (SP8, DMi8) and DFC 365 FX Kamera (1392 x 1040, 1.4 Megapixel)</td>
		</tr>
		<tr>
			<td>Di Potassium hydrogen phosphate (KH2PO4)</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>3904.1</td>
			<td>used for PBS buffer</td>
		</tr>
		<tr>
			<td>Drying material</td>
			<td>Merck</td>
			<td>101969</td>
			<td>orange silica beads for drying used in a glass bottle with a tubing adaptor</td>
		</tr>
		<tr>
			<td>ECGM supplement Mix</td>
			<td>Promocell</td>
			<td>C-39215</td>
			<td>supplement mix for ECGM -medium, required for precultivation of endothelial cells: 0.02 mL/mL Fetal calf serum, 0.004 mL/ mL endothelial cell growth supplement, 0.1 ng / mL epidermal growth factor, 1 ng / mL basic fibroblast growth factor, 90 &#181;g / mL heparin, 1 &#181;g / mL Hydrocortisone</td>
		</tr>
		<tr>
			<td>ECGMS</td>
			<td>Promocell</td>
			<td>C-22010</td>
			<td>ECGM supplemented with 5 % [w/w] FCS and 1 mM MgSO4 to increase cell adhesion</td>
		</tr>
		<tr>
			<td>Endothelial Cell growth medium (ECGM, ready to use)</td>
			<td>Promocell</td>
			<td>C-22010</td>
			<td>culture medium of HUVECs, already supplemented with all components of the supplement mix</td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>biochrome now Merck</td>
			<td>S 0415</td>
			<td>supplement for cell culture, used for infection analyses</td>
		</tr>
		<tr>
			<td>FITC-conjugated goat anti-human VWF antibody</td>
			<td>Abcam</td>
			<td>ab8822</td>
			<td>stock concentration: 10 mg/mL, for immunodetection of globular and multimerized VWF in flow</td>
		</tr>
		<tr>
			<td>Fluidic Unit</td>
			<td>ibidi</td>
			<td>10903</td>
			<td>fluidic unit for flow cultivation</td>
		</tr>
		<tr>
			<td>Gelatin (porcine)</td>
			<td>Sigma Aldrich</td>
			<td>G-1890-100g</td>
			<td>for precoating of microslide channel surface</td>
		</tr>
		<tr>
			<td>histamine dihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>H-7250-10MG</td>
			<td>for induction of VWF secretion from endothelial Weibel Palade Bodies</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cell (HUVEC)</td>
			<td>Promocell</td>
			<td>C-12203 Lot-Nr. 396Z042</td>
			<td>primary endothelial cells from pooled donor, stored crypcoserved in liquid nitrogen</td>
		</tr>
		<tr>
			<td>Human VWF-specific antibody derived from mouse (monoclonal)</td>
			<td>Santa Cruz</td>
			<td>sc73268</td>
			<td>stock concentration: 200 &#181;g/mL for immunostaining of VWF in microfluidic slide after PFA fixation</td>
		</tr>
		<tr>
			<td>Injection Port</td>
			<td>ibidi</td>
			<td>10820</td>
			<td>for injection of histamin or bacteria into the reservoir tubing during the flow circulation</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 35M</td>
			<td>inverse light microscope for control of eukaryotic cell detachement and cell counting using a 40 x water objective allowing 400 x magnification</td>
		</tr>
		<tr>
			<td>Luer-slides I0.4 (ibiTreat472microslides)</td>
			<td>ibidi</td>
			<td>80176</td>
			<td>physically modified slides for fludic cultivation (&#956;&#8211;Slide I0.4Luer with a channel hight of 0.4 mm, a channel volume of 100 &#956;l, a growth area of 2.5 cm and a coating area of 25.4 cm2) suitable for all kinds of flow assay, the physical treatment generates a hydrophilic and adhesive surface.</td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4, unhydrated)</td>
			<td>Sigma Aldrich</td>
			<td>M7506-500G</td>
			<td>For preparation of ECGMS medium</td>
		</tr>
		<tr>
			<td>Microfluidic Pump</td>
			<td>ibidi</td>
			<td>10905</td>
			<td>air pressure pump</td>
		</tr>
		<tr>
			<td>Neubauer cell counting chamber</td>
			<td>Karl Hecht GmbH&#38;Co KG</td>
			<td>40442002</td>
			<td>microscopic counting chamber for HUVECs</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td>for cross linking of samples</td>
		</tr>
		<tr>
			<td>Perfusion Set</td>
			<td>ibidi</td>
			<td>10964</td>
			<td>Perfusion Set Yellow/Green has a tubing diameter of 1.6 mm, a tube length of 50 cm, a total working volume of 13.6 mL, a dead tube volume of 2.8 mL and a reservoir size of 10 mL. combined with the &#181;-slide L0.4Luer, at 37&#176;C and a viscosity of 0.0072 dyn x s/cm2 a flow rate range of 3.8mL/min up to 33.9 mL/min and shear stress between 3.5 dyn/cm2 and 31.2 dyn/cm2 can be reached. with 50 cm lenght for microfluidic</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td>the solution was prepared using the following chemicals: 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4 , pH 7.4</td>
		</tr>
		<tr>
			<td>Plastic cuvettes</td>
			<td>Sarstedt</td>
			<td>67,741</td>
			<td>(2 x optic) for OD measurement at 600 nm</td>
		</tr>
		<tr>
			<td>Pneumococcus-specific antiserum</td>
			<td>Pineda</td>
			<td></td>
			<td>raised in rabbit using heat-inactivated Streptococcus pneumoniae NCTC10319 and D39, IgG-purified using proteinA-sepharose column.</td>
		</tr>
		<tr>
			<td>Polystyrene or Styrofoam plate</td>
			<td></td>
			<td></td>
			<td>this is a precaution step to avoid cold stress on the cells seeded in the channel slides. Any type of styrofoam such as packaging box-material can be used. The plate might by 0.5 cm thick and should have a size of 20 cm2.</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>6781.1</td>
			<td>used for PBS buffer</td>
		</tr>
		<tr>
			<td>Pump Control Software (PumpControl v1.5.4)</td>
			<td>ibidi</td>
			<td>v1.5.4</td>
			<td>Computer software for controlling the pressure pump, setting the flow conditions and start/end the flow</td>
		</tr>
		<tr>
			<td>reaction tubes 1.5/ 2.0mL</td>
			<td>Sarstedt</td>
			<td>72.706/ 72.695.500</td>
			<td>required for antibody dilutions</td>
		</tr>
		<tr>
			<td>reaction tubes with 50 mL volume</td>
			<td>Sarstedt</td>
			<td>6,25,48,004</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP-expressing pneumococci</td>
			<td>National Collection of Type Cultures, Public Health England</td>
			<td>10,319</td>
			<td>Streptococcus pneumoniae serotype 47 expressing RFP fused to ahistone-like protein integrated into the genome</td>
		</tr>
		<tr>
			<td>serological pipets 5, 10 mL</td>
			<td>Sarstedt</td>
			<td>86.1253.025/ 86.1254.025</td>
			<td>for pipeting larger volumes</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3, water free)</td>
			<td>Sigma Aldrich</td>
			<td>451614-25G</td>
			<td>for preparation of 100 mM Sodium Carbonate buffer</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate (NaH2PO4)</td>
			<td>Carl Roth GmbH + Co. KG, Karlsruhe</td>
			<td>P030.2</td>
			<td>used for PBS buffer</td>
		</tr>
		<tr>
			<td>Spectral Photometer Libra S22</td>
			<td>Biochrom</td>
			<td>80-2115-20</td>
			<td>measurement of optical density (OD) of bacterial suspension at 600 nm</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S0389-500G</td>
			<td>for preparation of blocking buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284-500ML</td>
			<td>Used in 0.1% end concentration diluted in dH20 for eukaryotic cell permeabilization after PFA fixation</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>oxoid</td>
			<td>LP0021</td>
			<td>bacteria are cultivated in THB supplement with 1% [w/w] yeast extract = complete bacterial cultivation medium THY</td>
		</tr>
	</tbody>
</table>

pneumococcus infection of primary human endothelial cells in constant flow

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.

Culturing primary HUVEC in a constant unidirectional flow results in the formation of a confluent and tightly packed cell layer that promotes the generation of cellular WPBs filled with the mechanosensitive VWF13,14. This protocol describes the use of an air pressure pump-based, pulseless recirculation system for infection analysis that requires mimicking the shear stress situation in the human blood flow.
This system enables a defined...

Watch this Scientific Journal Video about Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow at JoVE.com

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

The use of this endothelial cell culture system enables assimilation of the situation in the blood flow. And with this situation, we can analyze bacterial infection processes also including the identification of bacterial vectors and also of cell vectors which are involved in this process. This technique paves the way to explore the impact of bacterial infections on the vascular integrity, also including the impact on morphogenesis mechanisms, on inflammation responses and immune regulation.This technique requires some special handling of the endothelial cells and therefore a visual description is better than just a verbal explanation. This method provides insights into several other areas of research correlated with perfused vascular systems such as endothelial cell differentiation, wound healing, tumor genesis and angiogenesis. One of the key steps is standardized cell cultivation as well as accurate handling of primary endothelial cells.This technique requires some special cell culture handling which couldn't be clear by just verbal explanation. To begin, work in a sterile environment using a clean bench. Use a pipette to inject 100 microliters of a 2%sterile filtered porcine gelatin PBS solution into a single reservoir of a temperature equilibrated channel slide.Place the gelatin-coated channel slide on a thin polystyrene or styrofoam plate to prevent a drop in the slide temperature with a one milliliter Luer syringe to add 100 microliters of the four times 10 to the fifth per milliliter HUVEC suspension into the slide to seed and cultivate the HUVEC. Incubate the channel slide with the HUVEC for 60 minutes at 37 degrees Celsius and 5%carbon dioxide. After that, fill up each medium reservoir at both ends of the channel slide with 60 microliters of ECGMS medium.Incubate for another one hour at 37 degrees Celsius and 5%carbon dioxide. Next, to adjust the microfluidic pump, first connect the equilibrated perfusion set to the pump unit, fill with 13.6 milliliters of ECGMS medium and start the pump control software. In the menu of the fluidic unit setup, select the adequate perfusion set and type of chamber slide using the scroll down windows.Choose 0.007 dyne second per square centimeter in the software for medium viscosity. Outside of the incubator, connect a glass bottle filled with drying silica beads to the air pressure tubing. Select flow parameters in the software menu, set the pressure to 40 millibar and flush the pump tubes with the liquid medium by starting the continuous medium flow.We ensure a tight cell attachment by using subconfluently grown HUVECs and additionally focus on getting air bubbles out of the system as well as using independent power supply for the pump system. Tap at the tubing connections to remove air bubbles and ensure that no air bubbles are circulating in the pump system. For connecting the channel slide, stop the flow circulation in the pump control software and hold the medium flow and the perfusion tubing by clamping the tubes near the Luer connection.Connect the channel slide thereby avoiding air bubbles. In the pump control software advanced tab, use the software tool cycle creator to program the desired shear stress cycles for flow cultivation. Program the shear stress levels for flow circulation and start long-term perfusion.Start with five dyne per square centimeter which corresponds to a flow rate of 5.44 milliliters per minute for 30 minutes followed by continuous flow at shear stress of 10 dyne per square centimeter which corresponds to a flow rate 10.85 milliliters per minute. Control balanced reservoir pumping. Place the fluidic unit with the connected channel slide in an incubator at 37 degrees Celsius and 5%carbon dioxide.Start the microscope software control and adjust the principle settings for the fluorescence microscopic monitoring by selecting the appropriate filter settings. For microscopic visualization, place the fluidic unit into the 37 degrees Celsius heating chamber. Place the channel slide on the stage of a prewarmed microscope.Control the cell morphology and the integrity of the HUVEC layer prior to injection of histamine and bacteria to the flow circulation. Maintain the flow setting. Induce the release of VWF from endothelial viable Palade bodies by injecting 136 microliters of a 100 millimolar histamine stock solution through an injection port into the ECGMS medium circulating in the perfusion tubing.For immunofluorescence detection of multimerized VWF strings, inject 20 micrograms of a VWF specific FITC conjugated antibody in a volume of 200 microliters of PBS into the circulating 13.6 milliliters of ECGMS medium. To quantify Pneumococcal attachment to the VWF strings generated on HUVEC cell surfaces, inject 1.35 times 10 to the eighth CFU per milliliter RFP expressing Pneumococci in a maximum volume of one milliliter into the ECGMS medium using the injection port. Select a 63X oil immersion objective for microscope magnification and adjust the fluorescence filter settings in the microscope software to the RFP channel with a 540 nanometer detection filter for detection of RFP expressing Pneumococci.For quantification of bacterial attachment to the VWF strings, create snapshots of Z stacks of at least 30 representative field views each containing approximately 10 morphologically intact HUVEC and count the amount of Pneumococci. To evaluate bacterial attachment after fixation prior to immunofluorescent staining, stop the flow, remove 10 milliliters of ECGMS medium from the pump reservoirs, and add 10 milliliters of PBS supplemented with 5%paraformaldehyde and proceed according to the manuscript. In this protocol, the principle experimental steps begin with a pre-cultivation of primary endothelial cells to subconfluency in cell culture flasks.Cells are then seeded into a gelatin-coated channel slide. Bacteria are grown on agar plates followed by cultivation in a complex liquid medium to mid log phase. For microfluidic cell culture, a channel slide with endothelial cells is connected to the perfusion tubes of a fluidic unit of the pump system and subjected to constant flow for cell differentiation.The generation of the VWF strings was induced by histamine injection at a shear stress of 10 dyne second per square centimeter and microscopically monitored by immunofluorescence detection using FITC-labeled VWF specific antibodies. After injection of red fluorescence protein expressing bacteria to the circulating medium, Pneumococcus attachment to the VWF strings was microscopically visualized in real time by fluorescence emission at 450 nanometers. After fixation of the cells using PFA, differential immunofluorescence staining provides the visualization of bacterial attachment at specific infection time points.The most important step of this procedure is to ensure a tight cell attachment on the slide surface by adjusting physiological shear stress levels and avoiding extreme pump pressure fluctuations. In addition to the microscopic visualization, this technique can be applied to study pharmacological effects of new anti-infective and also to analyze long-term consequences of the vascular infection processes. This pump system perfuses bacterial pathogens by air pressure to prevent any transmission via aerosols.It is recommended to follow the biosafety precautions.

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