Micro-Parallel Plate Flow Chamber Model for Studying Bacterial Adhesion to Human Endothelial Cells

الملخص

Source: Claes, J., et al, In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions. J. Vis. Exp. (2015).

This video demonstrates a method for studying host-pathogen interactions using a micro-parallel plate flow chamber seeded with human primary endothelial cells. Following endothelial cell activation by an ionophore and perfusion with fluorescently labeled bacteria, the cells release von Willebrand factor (VWF), which binds bacterial adhesins under shear flow conditions. Fluorescence microscopy is then used to visualize and quantify adherent bacteria on the cell surface.

البروتوكول

1. In Vitro Perfusion Experiments

1. Coating of Glass Coverslips
   1. Dilute von Willebrand factor (VWF) (Haemate P, stock concentration 2,400 µg/ml) in laboratory-grade water (deionized distilled) to a final concentration of 50 µg/ml.
   2. Dilute collagen in isotonic glucose solution (SKF solution, pH 2.7-2.9, as supplied by the manufacturer) to a final concentration of 160 µg/ml.
   3. Coat glass coverslips (24 × 50 mm) with VWF or collagen by dropping 200 µl of the coating on parafilm and placing the coverslip on top of the droplet. The dro....

النتائج

Figure 1. A schematic representation of the in vitro flow model. The in vitro flow model is a multifunctional model, which allows the study of different shear-dependent mechanisms, such as bacterial adhes.......

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
Brain Heart Infusion (BHI)	BD Plastipak	237500
Tryptic Soy Broth (TSB)	Oxoid	CM0129
Phosphate Buffered Saline (PBS)	Invitrogen	14190-169	D-PBS
5(6)-carboxy-fluorescein N-hydroxysuccinimidyl ester	Sigma-Aldrich	21878-25MG-F	fluorescent labeling
Bovine Serum Albumin Fraction V (BSA)	Roch	10 735 086 001
Haemate-P	CSL Behring	PL 15036/0010	VWF
Horm collagen	Takeda	10500	collagen
1-well PCA cell culture chambers	Sarstedt	94.6140.102	plastic slips