eoe sub articles

EoE Sub Articles

1. Analysis of Vitronectin, Vn as a Bacterial Surface Protein-Ligand
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	<li>Detection of Vn-binding at the bacterial surface using flow cytometry 
	NOTE: In flow cytometry, we used side scatter and forward scatter to gate positive events. To examine the interactions with Vn, Haemophilus influenzae&#160;type f, Hif clinical isolates (n=10)&#160;were selected together with&#160;E. coli&#160;BL21 (DE3) as a negative control (Figure 1A</st...

detection of vitronectin binding to bacterial surfaces using flow cytometry

Source: Singh, B. et al., <a href="https://app.jove.com/v/54653">Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance.</a>&#160;J. Vis. Exp.&#160;(2018)
This video demonstrates the detection of vitronectin&#39;s interaction with bacterial surface proteins. The flow-cytometry analysis measures the fluorescence signal of fluorescently labeled antibodies bound to vitronectin, confirming its presence at the bacterial surface. This technique provides insights into host-pathogen interactions and potential bacterial infection therapies.

<img alt="Figure 1" src="/files/ftp_upload/21806/21806_Figure1.jpg" /> 
Figure 1:&#160;Haemophilus influenzae&#160;serotype f binds Vn via surface-expressed PH.&#160;(A) Flow cytometry results showing binding of Vn to cells of Hif clinical isolates. Each clinical isolate (5 x 106&#160;CFU) was incubated with 250 nM human Vn. The bound ligand was detected using sheep anti-Vn pAbs and FITC-conjugated donkey anti-she...

Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

1. Analysis of Vitronectin, Vn as a Bacterial Surface Protein-Ligand

	Detection of Vn-binding at the bacterial surface using flow cytometry
	NOTE: In ...

Take flow cytometer tubes containing wild-type and mutant strains of Haemophilus influenzae, type f.
Pipette a buffer containing blocking proteins and vitronectin. Incubate.
The blocking proteins reduce non-specific interactions on the bacterial surface, while vitronectin molecules bind to protein H, a vitronectin-binding protein, on wild-type bacterial surfaces. In the mutant strain, vitronectin does not bind due to the absence of protein H.
Centrifuge. Remove unbound vitronectin and blocking proteins.
Add a buffer containing primary anti-vitronectin antibodies, which specifically bind to vitronectin attached to the wild-type bacterial surfaces.
Next, introduce fluorophore-conjugated secondary antibodies, which bind to the primary antibodies on vitronectin and facilitate vitronectin detection.
Centrifuge. Remove unbound secondary antibodies. Resuspend bacteria in the buffer.
Perform flow cytometry to determine vitronectin binding to the bacterial surface.
Compare the fluorescence signals of wild-type and mutant strains. A shift in the fluorescence signal in wild-type bacteria confirms vitronectin binding to the bacterial surface.