a. Scanned image of the XTT reduction assay for quantitation of biofilms. b. Quantitation of biofilms by XTT reduction assay. All experiments were done in triplicate with three technical repeats on separate days with similar results and shown as a representative image. RPMI 1640/HS

vs. RPMI 1640, **p < 0.01. To confirm the hypothesis that this effect was not specific to strain ATCC90028, we tested three unrelated clinical strains and found that HS also had the same effect on all three clinical strains C59 wnt mouse (data not shown). Characterization of the inhibitory components To further investigate the component(s) of serum that affect the adhesion of C. albicans, we heated the serum at 56°C for 30 min. This heat treatment did not abrogate the inhibitory activity. Heat-inactivated serum still inhibited biofilms in a dose-dependent manner (Figure 2A). At a concentration of 3%, heat-inactivated HS significantly inhibited biofilm formation (p < 0.001), and with increasing HS concentrations, the effect of HS

on biofilm formation became more pronounced. To eliminate the possibility that a heat stable protein was responsible for the biofilm inhibition, proteinase K was used to degrade proteins in the HS, but this also did not affect the ability of serum to inhibit biofilm formation (Figure 2B). CT99021 chemical structure Biofilm formation was significantly reduced in proteinase K-treated Phosphatidylinositol diacylglycerol-lyase serum compared with the control group (all p < 0.001). At a concentration of 3%, proteinase K-treated HS significantly inhibited biofilm formation (p < 0.001), and with increasing HS concentrations, the effect of HS on biofilm formation became more pronounced. The results were similar in all four C. albicans strains

(data not shown). Figure 2 The component(s) of serum inhibit C. albicans biofilm formation. A) Biofilm formation of C. albicans ATCC90028 was examined in the presence of different concentrations of heat-inactivated human serum for 24 h at 37°C. a. Scanned image of the XTT reduction assay for quantitation of biofilms. b. Quantitation of biofilms by XTT reduction assay. B) Biofilm formation of C. albicans ATCC90028 was examined in the presence of different concentrations of proteinase K-treated human serum for 24 h at 37°C. (a. Scanned image of the XTT reduction assay for quantitation of biofilms. b. Quantitation of biofilms by XTT reduction assay.) All experiments were done in triplicate with three technical repeats on separate days with similar results. RPMI 1640/HS vs. RPMI 1640, **p < 0.01. Effect of human serum on planktonic growth of C. albicans To confirm that inhibition of biofilm formation was not due solely to growth inhibition, the effect of HS on the planktonic growth of C. albicans was investigated.