Operetta microscope

Microscopy was done using a PerkinElmer Operetta microscope as established previously^{9 (link)}. Four sites were acquired per well. Laser-based autofocus was performed at each imaging position. Images of two channels (DAPI and GFP) were collected using a 60 × high-NA objective to visualize the cell and the aggregation states of the proteins, respectively. At every site and every fluorescent channel 5 images were taken at different z positions with 0.5 μm shifts. These images were used for a perfect focus algorithm. Cellular properties of about 1000 cells of each expressing strain were extracted from the images, including the localization of the GFP signal within the cell.

1280 approved drugs were analyzed for their impact on Sbds^R126T/R126T cells growth. Cells were seeded using a Multidrop Combi (Thermo Scientific) in 384 well plates (Perkin Elmer Cell Carrier, collagen coated) at a density of 4000 cells/well. After 24 hours the drugs were supplemented using a Hamilton Starlet liquid handler at a final concentration of 10 μM and cells were treated for 30 hours. The reference compound for toxicity was 5 μM Trichostatin A. Cytotoxicity was than evaluated by measuring in vivo nuclear shrinkage and loss of cells using Hoechst33342, and cell membrane disruption using the cell-impermeant nuclear dye BOBO-3. Briefly, following incubation with the drugs diluted in 50 μl growth medium, 25 μl of a dye cocktail containing 3 μM Hoechst33342 and 2.25 μM BOBO-3 were added and incubation was continued for further 45 min at 37°C, 5% CO₂. Images were then immediately acquired and analyzed using the Operetta microscope (Perkin Elmer). For the validation of hits, 6-point dose-responses for each compound in duplicate were used (ranging from 30uM to 0.12uM). The number of dead cells/total number of cells was the readout of the assay, where dead cells correspond to nuclei stained with BOBO-3 and the total number of cells correspond to the nuclei stained with Hoechst33342.