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.
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