, 2009). Dilutions of the stock solution were then used in the assays, to yield a final concentration of 192 μg mL−1, 384 μg mL−1 of extract in samples. The concentration of methanol in a sample was never >0.8% and it had no visible effect on the cell lines. The extract was assayed in a susceptibility test according to the M27-A2 method of NCCLS (National Committee Roxadustat ic50 for Clinical Laboratory Standards, 2002). None of the tested concentrations influenced C. albicans growth. Caco-2 and Intestin 407 cells were obtained from the Polish Academy of Science Culture Collection (Wrocław). Both lines were cultivated at 37 °C in 5% CO2 in MEM+GlutaMAX™-I containing Earle’s

and 25 mM HEPES (Gibco) and 1% antibiotic/antimycotic solution (Gibco) supplemented with heat-inactivated fetal bovine serum (FBS, Gibco) at a final concentration of 20% for Caco-2 and 10% for Intestin 407. To obtain a fully confluent and enterocyte-like morphology in the case of the Caco-2 monolayer, Caco-2 and Intestin 407 cells were grown for 21 and 2–3 days, respectively. Alpelisib datasheet For the adhesion experiment, Caco-2 and Intestin 407 cells were seeded onto 96-well tissue microplates (Nunc) at a density of 2.0–2.5 × 104 cells per well at a final volume of 100 μL. Cells were grown

to ∼85–95% confluence with media refreshment every 2nd day. Subsequent, cells were washed to rinse out antibiotics and resuspended in the same medium supplemented with 2% FBS without antibiotics. Subsequent experiments were set up with the addition of: (1) C. albicans, (2) C. albicans with various concentrations of S. boulardii extract, and (3) C. albicans and S. boulardii at ratios of 1 : 1 (corresponding to 2 × 106 CFU mL−1 for both strains) and 1 : 10 (corresponding to 2 × 106 and 2 × 107 CFU mL−1 for C. albicans and S. boulardii, respectively). Control with S. boulardii extract contained 0.8%

methanol. The adhesion test was carried out for 3 h at 37 °C. After incubation, the wells were gently washed with PBS and cells were fixed with 4%p-formaldehyde. For a out quantitative assessment of binding, cells were stained with 0.5% crystal violet (Freshney, 2002; Noverr & Huffnagle, 2004). The OD595 nm was read in a spectrophotometer (Asys UVM 340, Biogenet). Results are expressed as the percent of C. albicans adhesion inhibition with respect to 100% adhesion of C. albicans to the relevant cell line in the control well. Experiments were repeated eight times, with six repetitions in each. For microscopic analysis, plates stained with 0.5% crystal violet were observed under the inverted microscope CKX41 (Olympus) using × 40 objective and photographed using an ARTCAM-300MI camera. For assessment of the cytokine mRNA response of Caco-2 line, cells were typically cultured in 6 mL standard medium supplemented with 10 mM butyric acid in 40-mL flasks (Nunc), as described previously (Saegusa et al., 2004).