Flow cytometry was used to analyze the binding of the human anti-F1 and anti-V antigen IgG1s to Y. pestis pgmwhole-cells. Y. pestis pgmwas first streaked onto a sheep-blood agar plate and grown at room temperature (RT) for 3 to 4 days until colonies were readily visible. A single colony was picked and inoculated into 10 ml of Heart Infusion Broth (Remel, Lenexa, KS) containing 0.2% xylose and 2.5 mM CaCl 2 , and cells grown overnight (o/n) at RT with rigorous shaking. The next day, an aliquot of the bacteria culture was shifted to 37uC, and the other remained at RT, and the cultures were allowed to continue for another 3 hours before the bacteria were collected and suspended in PBS. Ten mL of bacterial cells was mixed with 90 mL of Fc Block solution [PBS with 1% FCS and 10 mg/ml FcBlock (BD Bioscience)] to achieve a final density of 1610 7 cells/ml. Human or mouse IgGs were added to the bacterial cell suspension to a final concentration of 10 mg/ml. After incubating at 4uC for 30 min, the bacterial cells were collect by centrifugation and suspended in 1 ml of PBS, and cells washed twice. The bacterial cells were then suspended in the same Fc Block solution, and secondary antibodies, which included either goat anti-mouse IgG-FITC (Pierce, Rockford, IL) or goat anti-human IgG-FITC (Southern Biotech, Birmingham, AL) were added to the cells at a dilution of 1:100. After 30 min at 4uC, the cells were then washed three times with PBS and subjected immediately to FACS analysis using a FACSCalibur (BD Bioscience, San Diego, CA), after the cells were fixed with 4% paraformaldehyde. For detection of antibody binding to whole-cells by immunofluorescent microscopy, the growth of Y. pestis pgmand the sample preparation for binding by human or mouse anti-F1 or anti-V mAbs was identical as that for the FACS analysis, except a Nikon T-2000 fluorescent microscope (Nikon Instruments Inc., Melville, NY) was used for detection.