Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

Published: November 30, 2023

Abstract

Source: Singh, B. et al., Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance. J. Vis. Exp. (2018)

This video demonstrates the detection of vitronectin'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.

Protocol

1. Analysis of Vitronectin, Vn as a Bacterial Surface Protein-Ligand 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 type f, Hif clinical isolates (n=10) were selected together with E. coli BL21 (DE3) as a negative control (Figure 1A</st…

Representative Results

Figure 1: Haemophilus influenzae serotype f binds Vn via surface-expressed PH. (A) Flow cytometry results showing binding of Vn to cells of Hif clinical isolates. Each clinical isolate (5 x 106 CFU) was incubated with 250 nM human Vn. The bound ligand was detected using sheep anti-Vn pAbs and FITC-conjugated donkey anti-she…

Disclosures

The authors have nothing to disclose.

Materials

5 mL polystyrene round-bottom tube  BD Falcon 60819-138 12 × 75 mm style
Bovine Serum Albumins (BSA) Sigma-Aldrich A2058 Suitable for cell culture
Flow cytometer  BD Biosciences 651154 Cell analysis grade for research applications 
Vitronectin (Vn) from human plasma Sigma-Aldrich V8379-50UG Cell culture grade
E. coli host (E. Coli BL21) Novagen 69450-3 Protein expression host
FITC-conjugated donkey anti-sheep antibodies  AbD Serotec STAR88F Polyclonal
Sheep anti-human Vn antibodies AbD Serotec AHP396 Polyclonal

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Cite This Article
Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry. J. Vis. Exp. (Pending Publication), e21806, doi: (2023).

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