Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

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…

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