The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.
The implication of this technique extends towards potential therapy of neurodegenerative diseases, such as ALS, because it proposes a simple method that allow to develop cellular assay and perform screening for small molecule moderators. To start virus production, prior to plasmid transfection split HEK-293 T-cells at 30 to 40%confluence in a 10 centimeter tissue culture dish and 10 milliliters of cell culture medium. Incubate the culture plate in a 5%carbon dioxide incubator at 37 degrees Celsius overnight.
On the following day, prepare DNA transfection solution with packaging plasmids lentiviral vector, 125 microliters of one molar calcium chloride and add distilled water to adjust the volume to 500 microliters. Gently add 500 microliters of 0.05 molar HEPS buffer and incubate for 10 minutes at room temperature. After that, replace HEK-293 T-cell medium with 10 milliliters pre-warmed antibiotic-free fresh culture medium mixed with one milliliter of DNA transfection solution.
Incubate at 37 degrees Celsius and 5%carbon dioxide incubator over night. Replace cell supernatent with fresh culture medium on the following day and incubate again overnight. Finally, on the following day harvest the supernatent and transfer to a 15 milliliter tube.
To remove the dead cells and debris, centrifuge the tube at 500 times g for five minutes. To further purify the supernatent, pass it through a 0.45 micrometer filter using a 60-milliliter syringe. Then, immediately dispense the filtered supernatent into 300-microliter single use aliquots.
To prepare the cells for transduction, split them to be infected at 60%confluence in a six-well tissue culture plate in two milliliters of DMAM supplemented with 10%FBS. Incubate the cells in a 37-degree Celsius incubator at 5%carbon dioxide overnight. On the following day, thaw one aliquot of the frozen lentivirus on ice for each use.
Meanwhile, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, dilute it in DMEM in new 1.5 milliliter micro-centrifuge tubes. Then adjust the volume to one milliliter with reduced serum medium.
Next, add two microliters of polybrene to one milliliter of virus per medium and mix well by pipetting. Add one milliliter of this mixture to the cells and incubate at 37 degrees Celsius for 24 hours. After incubation, remove the virus containing medium and replace it with normal DMEM supplemented with 10%FBS and return the cells to the incubator.
Monitor the cell growth and change the medium every two days When the cells reach confluence, expand each well of the six-well dish into a 10-centimeter tissue culture plate. Move the plates to the incubator. Finally, save 1.5 times 10 to the 4th cells per well and 50 microliters of DMEM on 384 well assay plates.
Then check the expression level of the YFP-tagged protein under the microscope. If the expression shows signal-to-noise ratio equal or greater than three, prepare cell stock for the corresponding cell line. For the compound transfer, on a 384-well storage polypropylene plate, add serial dilutions of compounds by the standard one to three dilution series.
Then, using a liquid handler equipped with a 384-capillary head, distribute 0.25 microliters of proteasome inhibitors on empty, flat-bottomed black 384-well assay plates. Then seed 1.5 times 10 to the 4th 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 20 times objective in the correct plate type.
To allow proper focus with different plate types, make sure that the caller is set to the correct value on the objective. Next, select exposure one. Set focus height to zero micrometers, then select focus.
Once it is focused, expose camera one. To optimize the exposure plane, adjust the focus height and click on take height. Change exposure times and laser power for a maximum pixel intensity of approximately 3000 and save exposure parameters.
Select experiment definition tab, create a layout and sublayout. Next, drag and drop the relevant layout exposure, reference image, skew crop file, and sublayout. Then, save the experiment.
Finally, select automatic experiment tab and acquire images. For two-channel images, first select the software algorithm to segment the primary objects, such as nuclei, cytosol, and aggregates. First, distribute 0.25 microliters of proteasome inhibitor dissolved in 100%DMSO or DMSO on 384-well flat-bottomed plates.
On the same plate, manually seed 1.5 times 10 to the 4th cells per well in 50-microliters of DMEM. Then set up the microscope system environmental control unit to 37 degrees Celsius and 5%carbon dioxide. Operate the microscope in wide-field florescence mode and as software settings, choose LWD 20 times objective non-confocal mode, four fields per well, and capture interval of one hour.
Then, select the appropriate algorithm to segment the primary object such as cytosol and aggregates. Adjust background threshold and contrast parameters if required. The tested cell lines transduced with SOD-1 wild type and SOD-1 A4V mutant remained healthy, showed high expression levels, and longterm wide phi-labeling irrespective of the SOD form.
Treatment with proteasome inhibitor ALLN for 24 hours promoted the accumulation of SOD-1 A4V YFP aggregates in HEK-293 cells but not in SOD-1 Wild Type YFP transduced cells. Furthermore, there were very low levels of aggregation in U2 OS and SH-SY5Y cell lines SOD-1 A4V YFP transduced with the same vectors. After seeding the cells in the presence or absence of ALLN, they were monitored for aggregate formation for a period of 50 hours.
ALLN aggregate formation reached a plateau at 24 hours and remained stable over the next 12 hours. Furthermore, cell density significantly decreased after 28 hours as a result of the cytotoxic effect of ALLN, while cell number increased in the DMSO-treated negative control. SOD-1 A4V YFP cells cultured in the presence of proteasome inhibitors for 24 hours were stained with Hoechst solution.
The relative toxicity for three different proteasome inhibitors was quatified by measuring the number of adherent cell lines remaining in the well labeled with Hoechst dye. The total cell number decreased in response to increased dose of protease inhibitors. Inversely, the percentage of cells expressing SOD-1 A4V aggregates increased with the concentration of protease inhibitor.
When doing this process it is important to remember that the cell quality will drastically influence your result. So it's important to determine that the cells are microplasma free and are healthy all along the steps. Following this process, another imaging method can be used in order to answer additional questions regarding the degradation of protein or the interaction of the protein with others using resonance energy transfer.
After watching this video, you should have a good understanding of how to generate the tools to test the fate of protein in multiple living cells using high-content image analysis. So this method can provide insight into protein aggregation as it occurs in simple cellular models. It can also be applied to other systems such as neuron differentiated from patient specific induced pluripotent stem cells.
