Evaluating Pneumococcal Attachment to Primary Human Endothelial Cells Under Constant Media Flow

Samenvatting

Source: Jagau, H. et al. Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow. J. Vis. Exp. (2019)

This video demonstrates the real-time visualization of pneumococcal attachment to Von Willebrand Factor (VWF) strings on endothelial cells under constant media flow conditions. It outlines the steps for stimulating VWF release, labeling the strings, and quantifying bacterial binding.

Protocol

1. Induction of Von Willebrand Factor (VWF)-release and Visualization of Multimerized VWF Strings

1. Maintain the flow setting because a shear stress of 10 dyn/cm² is required to trigger the multimerization of VWF to long strings of up to 200 MDa. Induce the release of VWF from endothelial WPB by injecting 136 µL of a 100 mM histamine stock solution into the endothelial cell growth medium with supplement mix (ECGMS)-medium circulating in the perfusion tubing using an injection port. The final concentration of histamine in the flow medium will be 1 mM. If no injection port is available, the histamine can be added alternatively by pipetting into the medium of the pump reservoirs.
2. For immunofluorescence detection of multimerized VWF strings, stop the flow when a balanced medium level in the reservoirs is reached and inject 20 µg of a VWF-specific fluorescein isothiocyanate (FITC)-conjugated antibody in a volume of 200 µL phosphate-buffered saline (PBS) (pH 7.4) into the circulating 13.6 mL of ECGMS-medium using an injection port. If no injection port is available, the antibody can be added alternatively by pipetting into the medium of the pump reservoirs. This results in a final antibody concentration of 1.3 µg/mL.
3. For microscopic scanning of several fields of view in a short time, use the fluorescence unit of the microscope with a Xenon fluorescence device at 30% power and an epifluorescence camera. Monitor the shape and morphology of the human umbilical vein endothelial cell (HUVEC) layer with the bright field mode to select representative cells suitable for the VWF-strings visualization.
4. For visualization of green fluorescent VWF strings, select a 63x/1.40 oil objective and a 470 nm detection filter in the fluorescence unit menu of the microscope software (LasX). Create snapshots of Z-stacks of at least 50 representative field views, each containing approximately 10 morphologically intact HUVEC. For quantification of the green fluorescent VWF strings at different time points, scan several fields of view.

2. Microscopic Evaluation of Bacterial Attachment to VWF-strings in Flow in Real Time

1. Quantify pneumococcal attachment to the VWF-strings generated on HUVEC cell surfaces via immunofluorescence detection.
   1. Hold the flow and inject 1.35 x 10⁸ CFU/mL red fluorescence protein (RFP)-expressing pneumococci in a maximum volume of 1 mL into the ECGMS medium using the injection port. Alternatively, pipette the bacteria into the medium in the pump reservoir. Restart the shear stress at 10 dyn/cm² to let the bacteria circulate within the pump system.
   2. Select a 63x oil-immersion objective for microscope magnification and adjust the fluorescence filter settings in the microscope software to the RFP-channel (540 nm detection filter) for the detection of RFP-expressing pneumococci.
   3. For quantification of bacterial attachment to the VWF strings, stop the flow and create snapshots of Z-stacks of at least 30 representative field views, each containing approximately 10 morphologically intact HUVEC, and count the number of pneumococci.
   4. Use the ANOVA one-factorial statistics algorithm in order to evaluate the data, followed by a post hoc two-tailed unpaired sample test for detailed statistical comparison. P values of <0.05 were considered statistically significant.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Clamp for perfusion tubing	Ibidi	10821	For holding the liquid in the tube bevor connecting the slide to the pump system
Computer	Dell	Latitude 3440	Comuter with pressure pump software
Confocal Laser Scanning Microscope (CLSM)	Leica	DMi8	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)
Di Potassium hydrogen phosphate (KH₂PO₄)	Carl Roth GmbH + Co. KG, Karlsruhe	3904.1	Used for PBS buffer
Drying material	Merck	101969	Orange silica beads for drying used in a glass bottle with a tubing adaptor
ECGM supplement Mix	Promocell	C-39215	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 µg / mL heparin, 1 µg / mL Hydrocortisone
ECGMS	Promocell	C-22010	ECGM supplemented with 5 % [w/w] FCS and 1 mM magnesium sulfate to increase cell adhesion
Endothelial Cell growth medium (ECGM, ready to use)	Promocell	C-22010	Culture medium of HUVECs, already supplemented with all components of the supplement mix
Fetal Calf Serum (FCS)	Biochrome now Merck	S 0415	Supplement for cell culture, used for infection analyses
FITC-conjugated goat anti-human VWF antibody	Abcam	ab8822	Stock concentration: 10 mg/mL, for immunodetection of globular and multimerized VWF in flow
Fluidic Unit	Ibidi	10903	Fluidic unit for flow cultivation
Gelatin (porcine)	Sigma Aldrich	G-1890-100g	For precoating of microslide channel surface
Histamine dihydrochloride	Sigma Aldrich	H-7250-10MG	For induction of VWF secretion from endothelial Weibel Palade Bodies
Human Umbilical Vein Endothelial Cell (HUVEC)	Promocell	C-12203 Lot-Nr. 396Z042	Primary endothelial cells from pooled donor, stored crypcoserved in liquid nitrogen
Human VWF-specific antibody derived from mouse (monoclonal)	Santa Cruz	sc73268	Stock concentration: 200 µg/mL for immunostaining of VWF in microfluidic slide after PFA fixation
Injection Port	Ibidi	10820	For injection of histamin or bacteria into the reservoir tubing during the flow circulation
Luer-slides I0.4 (ibiTreat472microslides)	Ibidi	80176	Physically modified slides for fludic cultivation (μ–Slide I0.4Luer with a channel hight of 0.4 mm, a channel volume of 100 μl, 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.
Magnesium sulfate (MgSO₄, unhydrated)	Sigma Aldrich	M7506-500G	For preparation of ECGMS medium
Microfluidic Pump	Ibidi	10905	Air pressure pump
Perfusion Set	Ibidi	10964	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 µ-slide L0.4Luer, at 37°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 length for microfluidic
Phosphate-buffered saline (PBS)			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
Potassium chloride (KCl)	Carl Roth GmbH + Co. KG, Karlsruhe	6781.1	Used for PBS buffer
Pump Control Software (PumpControl v1.5.4)	Ibidi	v1.5.4	Computer software for controlling the pressure pump, setting the flow conditions and start/end the flow
RFP-expressing pneumococci	National Collection of Type Cultures, Public Health England	10,319	Streptococcus pneumoniae serotype 47 expressing RFP fused to a histone-like protein integrated into the genome