Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

For the compound transfer, on a 384-well storage polypropylene plate, add serial dilutions of compounds by the standard 1 to 3 dilution series. Then, using a liquid handler equipped with a 384-capillary head, distribute 0.25 microliters of proteasome inhibitors on empty flat-bottom black 384-well assay plates. Then, seed 1.5 x 10⁴ cells on these plates. Incubate at 37 degrees Celsius and 5% carbon dioxide for 24 hours.

Prior to imaging the cell line treated with proteasome inhibitors, add 10 microliters of pre-warmed DMEM with staining solution and incubate at room temperature for 10 minutes.

After staining, start the automated microscope operating software by selecting the "Microscope" tab. First, select the "Configuration" tab, and then, select 20X objective and the correct plate type.

To allow proper focus with different plate types, make sure that the "Collar" is set to the correct value on the objective.

Next, select 'Exposure 1,' set focus height to '0 micrometers,' then, select "Focus." Once it is focused, expose Camera 1.

To optimize the exposure plane, adjust the focus height and click on "Take Height." Change exposure times in laser power for a maximum pixel intensity of approximately 3,000, and save exposure parameters.

Select "Experiment Definition" tab. Create a layout and sublayout. Next, drag and drop the relevant layout, exposure, reference image, skewcrop file, and sublayout. Then, save the experiment.

Finally, select "Automatic Experiment" tab and acquire images. For 2-channel images, first select the software algorithm to segment the primary objects, such as nuclei, cytosol, and aggregates.