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

Removal of misfolded proteins in the cell is majorly regulated by the cellular ubiquitin-proteasome system, wherein the protein is first recognized and tagged by ubiquitin, a small regulatory protein, and trafficked to the proteasome, a multiprotein complex, for hydrolysis and clearance.

Impaired ubiquitin-proteosome system activity, a common occurrence in neurodegenerative diseases, can cause misfolded proteins to accumulate within the cells, forming insoluble protein aggregates with cytotoxic effects.

To visualize and quantify protein aggregation in vitro, obtain a suspension of transduced mammalian cells - expressing a fluorescent-labeled mutant protein that adopts a misfolded conformation with exposed hydrophobic residues - in suitable media.

Pipette increasing concentrations of a proteasome inhibitor to the wells of a black multi-well plate to minimize background fluorescence during imaging. Seed the mammalian cell suspension into the wells. Incubate to allow the cells to adhere to the plate bottom and proliferate.

Once inside, the proteasome inhibitor binds to the proteolytic sites of the proteasome and blocks its activity, causing the expressed mutant proteins to accumulate, which then interact via their exposed hydrophobic residues and form cytoplasmic aggregates, eventually leading to cell death.

Add a fluorescent DNA-binding dye to stain the cell nuclei. Image the plate. The fluorescent mutant protein aggregates appear dispersed within the unstained cytoplasm.

Wells containing higher proteasome inhibitor concentrations exhibit higher protein aggregate numbers to cell count ratio than wells with lower proteasome inhibitor concentrations.

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.