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