Adhesion assays MG-132 purchase Adhesion to polystyrene

was assessed as described previously [44] with slight modifications. An inoculum of 1.0 × 107 cells ml-1 in PBS or RPMI-1640 was prepared, of which 150 μl was added to individual wells of a 96-well microtiter plate. An identical set of each strain was dispensed into individual microcentrifuge tubes for use as the unwashed control, representing the total number of adherent and non-adherent cells. Following a 2-hr incubation period at 37°C, non-adherent cells were removed by washing the wells of the microtiter plate, while cells incubated in microcentrifuge tubes were pelleted at high speed on a benchtop centrifuge. The XTT-reduction assay [45] was then used to quantify the adhered and total amount of cells in each well and microcentrifuge tube, respectively. After incubation with the XTT-menadione substrate at 37°C for 3 hours, 75 μl of colored formazan was transferred to a fresh microtiter plate and absorbance was read at 490 nm. The adherence CBL-0137 capacity of each strain was calculated as the mean XTT-value of the washed cells relative to the mean XTT-value of the unwashed cells. Experiments were performed at least twice, each with 8 replicates per

strain tested. Statistical significance was assessed with an analysis of variance (ANOVA) between all strains compared using Prism 5.0 (GraphPad Software, Inc.) Analysis of fungal biofilms Formation of C. albicans biofilms and the XTT-reduction assay were performed Pyruvate dehydrogenase lipoamide kinase isozyme 1 as previously described [45]. As an alternative, Crystal Violet staining was used to estimate the biofilm mass selleck compound [45]. Briefly, biofilms were stained with 100 μl

of a 0.3% (w/v) Crystal Violet, 5% (v/v) isopropanol, 5% (v/v) methanol solution for 5 min, after which the wells were washed with water. The dye was then extracted from the biofilms using 100 μl ethanol, of which 75 μl was transferred to a clean microtiter plate and the absorbance was measured spectrophotometrically at 550 nm. Scanning electron microscopy was performed on biofilm samples formed on a coverslip (Thermanox, Nalge Nunc International) after 24 h incubation of a 0.5 ml inoculum containing 1.0 × 106 cells ml-1 according to previously described methods [46]. Non-adherent, planktonic cells were quantified from biofilm washes by plating serial dilutions of the pooled washes, from individual replicates, onto YPD agar plates, and colony forming units were determined following incubation at 30°C for 2 days. Macrophage killing assays The macrophage killing assay was performed as described by Palmer et al. [36]. The murine macrophage cell line used in this study, J774A.1, was purchased from ATCC and propagated in high glucose D-MEM supplemented with 10% FCS. Next, 2.0 × 105 J774A.1 cells in a volume of 0.75 ml were seeded in Lab-Tek Chambered Slides (Nalge-Nunc), incubated overnight at 37°C with 5% CO2. C.