Dc300f digital imaging system

High-throughput screening was performed at the Chemical Genetics Unit, Department of Microbiology, Research Center of Infection and Immunology, Li Ka Shing Faculty of Medicine, University of Hong Kong, on a library with 50,240 small molecules (ChemBridge, San Diego, CA, USA) to identify inhibitors of Y-H transition in C. albicans SC5314, as was previously described [29] (link). C. albicans SC5314 were seeded at 5×10³ cells per well in complete yeast peptone dextrose (YPD) (1% yeast extract, 2% peptone, 2% glucose/dextrose) supplemented with 20% heat-inactivated foetal bovine serum (Invitrogen, Carlsbad, CA, USA) in a total volume of 50 µl in 384-well microtitre plates. The small molecules were dissolved in dimethyl sulfoxide (DMSO) and were added to the wells at final concentration of 20 µg/ml, whereas the controls contained the same amount of DMSO but without small molecules. Assay plates were incubated at 37°C in 5% CO₂ for 12 h. Morphologies of the Candida were scored using a Leica DMIL inverted microscope equipped with DC300F digital imaging system (Leica Microsystems, Heidelberg, Germany). Small molecules with lower scores than that of the control (yeast-to-hypha transition inhibitors) were selected as primary hits.

MDCK cells were grown to 70 to 80% confluence on coverslips. Cells were propagated with influenza A/WSN/33 wild-type virus or mutant viruses at an MOI of 10 in fresh MEM containing 0.2% FBS and 1 μg/ml TPCK-treated trypsin. A total of 20 μM FA-6005 or DMSO was added at the indicated time points. Infections were stopped at the indicated time points by fixation with 500 μl of 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 min. After fixation, the cells were washed with PBS three times and permeabilized with 1 ml 70% ethanol at 4°C overnight. Custom Stellaris fluorescence in situ hybridization (FISH) probes for the detection of vRNA and mRNA of segment 5 of influenza virus were designed by utilizing the Stellaris FISH probe designer (Biosearch Technologies Inc., Petaluma, CA). The assay for simultaneous immunofluorescence (IF) microscopy and FISH was performed according to the manufacturer’s instructions (available at www.biosearchtech.com/stellarisprotocols). The slides were stored at 4°C overnight before being sealed with nail polish to prevent drying. The slides were observed and photographed using a Leica DMIL inverted microscope equipped with a DC300F digital imaging system (Leica Microsystems, Germany) or an LSM710 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).