2). For each strain, a series of 10-fold dilutions was then prepared in water over a range of concentrations from 10-1 to 10-5 relative to the initial culture. Spots of 5 μl from each dilution series were then plated on the indicated media and cultured at 30°C for 2 days. Individual colonies selleck were then counted and compared to the number of colonies observed from an untreated culture serially diluted at the beginning of the experiment. Several serial dilutions for each culture were done to ensure
that there were enough colonies to count for statistical significance and at least three independent cultures were tested and compared. Statistical significance was determined with the Student’s t-test. Note that after 3 hr, cells cultured in rich media without any cell death inducing agents were able to grow and to divide, hence the relative viability levels that are greater than 100%. In vivo detection of mitochondrial fragmentation, ROS accumulation, and caspase activation
Mitochondrial LY2874455 fragmentation check details was detected in S. boulardii cells using 10 nM Mitotracker Green (Molecular Probes), according to the manufacturer’s specifications. Intracellular ROS accumulation was examined after treatment with 5 μg/ml of dihydrorhodamine 123 (DHR123; Sigma Aldrich) . Activated caspase-like activity was detected in S. boulardii cells after treatment using a FLICA apoptosis detection kit (ImmunoChemistry Technologies, LLC) according to the manufacturer’s specifications [43, 44]. After exposure to reagents, S. boulardii
cells were harvested and examined using a Zeiss LSM 700 Confocal Laser Scanning Microscope. Fluorescence microscopy Cells were grown to mid-log phase in selective media and examined using a 63X oil-immersion objective and a pinhole size of 1 Airy Unit using a Zeiss LSM 700 Laser Confocal Microscope Images were captured and processed using the ZEN 2009 software package. Microarray experiments: array design Genomic sequences were obtained from the Saccharomyces Genome Database (downloaded from http://www.yeastgenome.org). These sequences were used to design a custom 8×15K array using the Agilent Non-specific serine/threonine protein kinase eArray software (http://earray.chem.agilent.com/). Each array had a minimum of 2 unique 60-mer probes designed against 6,612 open reading frames encoded by S. cerevisiae. This resulted in a total of 13,275 unique probes for each array, including Agilent hybridization controls. Microarray experiments: sample preparation, extraction, and purification S. boulardii cells were cultured in rich YPD media overnight, resuspended in fresh media, and allowed to reach exponential phase (an approximate OD600 value of 0.2). They were then resuspended in 45 mL of either water, for the control condition, or water containing 50 mM HCl for the experimental condition. The total number of cells in each experiment was 3 × 108, as measured with a spectrophotometer. After a 1.