Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

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.

To prepare for flow cytometry, resuspend the bacterial pellets with 50 microliters of blocking buffer containing 250 nanomolar vitronectin. Then, incubate the samples for 1 hour at room temperature without shaking. After incubation, pellet the bacteria by centrifugation at 3,500 x g for 5 minutes. Then wash the pellets three times using PBS and similar centrifugation steps.

Following wash, add 50 microliters of primary sheep anti-human vitronectin polyclonal antibodies to the bacterial pellet, at a 1 to 100 dilution in PBS/BSA. Incubate the suspension for 1 hour at room temperature. Then, wash the bacteria three times with PBS to remove unbound antibodies.

Next, add 50 microliters of PBS/BSA containing fluorescein isothiocyanate-conjugated donkey anti-sheep polyclonal antibodies to the pellet. Incubate at room temperature for 1 hour in the dark. Finally, after three washing steps, resuspend the bacterial pellet with 300 microliters of PBS, and analyze by flow cytometry.