In the first system (visual assay of stained cells), 2 × 107 cells of wild type and mp65Δ mutant strains were incubated with 105 BEC, and the adherence was expressed as the number of yeast cells adhering to 100 epithelial cells ± standard error. The mp65Δ mutant showed significantly reduced adherence to BEC (GSK1120212 datasheet Figures 5 A and 5B), whereas the revertant strain partially regained the ability to adhere to BEC, reaching a level similar to that of the wild type (C. albicans cells/BEC mean ± S.E.; wild type: 35 ± 2.0 vs. mp65Δ
mutant: 10 ± 1.5 vs. revertant: 25 ± 1.0; P < 0.05). In the second system, the number of C. albicans cells adhering to the surface and those remaining in the supernatant were analyzed in a time-dependent manner (Figure 5C). Adhesion of the wild type cells to Caco-2 cells
was rapid and efficient: after 30 min, about 65% of the www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html cells recovered had adhered to the Caco-2 cell monolayers, whereas only 35% were recovered from the supernatant. After 60 min the percentage of adhering cells increased to 75%, whereas the percentage of cells in the supernatant decreased to 25%. The mp65Δ mutant cells showed significantly reduced adhesion to the Caco-2 cells: after 30 and 60 min, the percentage of adhering cells was XAV 939 38% and 43% respectively, whereas the percentage of non-adhering cells was 62% and 57% respectively. In the revertant cells, the efficiency and kinetics of adhesion were similar to those in the wild type. Figure 5 Adhesion analysis of the mp65Δ mutant. (A) Adhesion of the mp65Δ mutant to BEC. filipin Representative fields randomly selected showing the interaction between yeast cells [wild type (wt), mp65Δ mutant (hom) and revertant (rev) strains] and BEC after 1 h of incubation at 37°C. The magnification bar corresponds to 100 μm. See the Methods section for more details. (B) Adhesion assay data. Histograms showing the adherence of the wild type (wt: black column),
mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains to BEC. The bars indicate the standard errors. Significant differences from wild type adhesion (P < 0.05) are indicated by asterisks. (C) Adhesion of the mp65Δ mutant to Caco-2 cell monolayers. Recovery of Candida cells [wild type (wt: black column), mp65Δ mutant (hom: grey column) and revertant (rev: white column) strains] at different time points (30 and 60 min) of incubation with Caco-2 cells. Adherent cells recovered after thorough washing out of the microplate (Panel 1). Non-adherent cells recovered from the supernatant (Panel 2). The results are the mean of 3 independent experiments. The bars indicate the standard deviations. To determine the effects of the absence of the MP65 gene on biofilm formation, we performed two quantitative in vitro assays (dry weight and XTT), which characterize total and living biomass, respectively.