Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

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.