Begin by launching the microscope operating software. Under the setup tab, choose the correct predefined plate type. Press eject to access the plate stage to position the plate and then press load to load the plate into the microscope.
Next, choose the 20 times air objective with a numerical aperture of 0.4. Adjust the correction collar of the 20x air objective for the plate thickness of the chosen plate type. Set the channel selection to Hoechst 33342, and adjust the imaging parameters for the Hoechst channel.
Test the chosen settings by selecting a representative well within the plate. On defined layout, select all plate wells and for fields. Then choose the corresponding folder under online jobs to transfer the data to the online analysis software.
The experiment settings are saved. Under the run experiment tab, the plate which is going to be imaged is named, and the imaging process is started. To begin image analysis, first launch the software, choose the images for analysis, and then click on the image analysis tab to start image segmentation.
Next, click on the plus sign in the input image tab to add a new building block. From the list, click on find nuclei. Choose the most appropriate segmentation method after inspecting the segmented objects on the image.
Adjust the detection parameters within the segmentation method. Click on define results and choose standard output as the method. Click on save analysis to database to save the analysis pipeline.
Under the batch analysis tab, click on the saved pipeline which will appear under the method option. Then select the data to be analyzed. Start the analysis by clicking on start analysis to generate the dataset for cell numbers.
The described cell-based assay relies on a reporter readout that indirectly reflects changes in the ATG4B activity, enabling the detection of both inhibitors and activators of ATG4B activity. The ratio of the ATG4B activity of each compound to its cell viability is used as a cutoff value for the identification of ATG4B inhibitors. Ratios greater than one indicate possible ATG4B activators and ratios less than one indicate possible ATG4B inhibitors.
