Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

The overall goal of this procedure is to detect protio pathic seeding activity from recombinant or biological sources, enabling the sensitive detection of protein, aggregates, and a sample. This method can help to answer key questions in the field of neurodegeneration. For example, how might we quantitatively evaluate changes in seeding activity following therapeutic intervention in animal models of disease?

The advantages of this technique include its high specificity towards low levels of protein aggregates. Its exquisite specificity, its ease of use, and its high throughput. Demonstrating this procedure today will be myself, along with co-author Brandon Holmes.

To prepare the biological seed material begin by weighing frozen brain tissue in a disposable whey boat. Then transfer the tissue into a conical tube, add ice cold homogenization buffer, such that the final solution is 10%to wait to volume and transfer the samples to a cold room on ice. Next, adjust a probe so indicator to the appropriate settings and rinse aside and bottom of the probe.

So indicator tip three times as indicated, wiping off the probe between each solution. When the probe is ready, submerge the tip into the homogenization buffer of one sample and start the sonicate delivering 25 total pulses. When the tissue is completely in suspension, spin down the homogenate and transfer the supinate to a clean tube.

Taking care not to disturb the palate. While numerous other homogenization techniques are available, probation is superior for isolating small amounts of protio pathic seeds from biological specimens. The mechanical dissociation enables consistent and efficient breakdown of tissue and subsequent seed extraction.

Then aliquot the supine and store the lysates at negative 80 degrees Celsius. To replay the biosensor cells under sterile conditions, rinse each cell line with warm PBS followed by a three minute incubation with trypsin EDTA. When the cells have detached, stop the reaction with warm culture medium and immediately transfer the cells into a conical tube.

Centrifuge the cells resus, suspending the pellet in fresh medium. Then count the cells and make a master mix cell dilution of 3, 500, 000 cells per 13 milliliters of medium. Using a multi-channel pipette, slowly transfer 130 microliters of the cells into each well of a flat bottom tissue culture treated 96 well plate taking care to place the pipette tips in the center of the wells without touching the bottom of the plate.

Once the cells have been plated, allow them to settle undisturbed for 10 minutes at room temperature and then incubate the plate overnight at 37 degrees Celsius, 5%carbon dioxide and greater than or equal to 80%relative humidity. The next day when the biosensor cells are 60 to 65%confluent, combine reduced serum medium liposome reagent, and biological seed source to make transduction complexes. Then gly pipette 20 microliters of transduction complex down the side of each individual biosensor well and return the treated cells to the incubator for another 24 to 48 hours before harvesting.

Untreated cells will show only diffuse fluorescence. However, cells treated with tau seed material will show bright aggregates After one to two days to harvest the cells, replace the cell culture medium with 50 microliters of tryin EDTA, stopping the reaction. After five minutes with 150 microliters of chilled culture, medium, immediately transfer the cells to a 96 well round bottom plate and pellet the cells, aspirate the S supernatants without disturbing the pellets, and then gently but thoroughly resus, suspend the cells in 50 microliters of 2%paraldehyde for 10 minutes.

Then spin down the cells again and resuspend the pellets in 200 microliters of chilled flow cytometry buffer as soon as possible. After fixation, load the plate onto a fret compatible flow cytometer and generate a side scatter area versus forward scatter area by variant plot. Next with cell line one running, adjust a side scatter and forward scatter voltages until the cell population is in the lower left quadrant.

Then using the polygon tool, define the cell population as gait P one. Now make a forward scatter height versus forward scatter area by variant plot and apply the P one gate. Next, define the single cells as gait P two and generate one histogram each.

For the C-F-P-Y-F-P and fret filters, apply gate P two to three different histograms, one each for the C-F-P-Y-F-P and fret filters for compensation. Spike in 30 microliters of the cell line one suspension into 200 microliters of the cell line two suspension. Then click the instrument settings icon followed by the compensation tab and check matrix to open the compensation table.

Generate a fret area versus CFP area by variate plot and apply gate P two. Then with cell line one and cell line two running, click the quadrant icon and draw quadrants such to the fluorescent negative and positive populations are separated by the lower two quadrants. Now create a statistics table that displays the median fluorescence intensity or MFI of fret for the LL three and LR three gates and adjust the fret parameter in the compensation matrix until the MFI of the fret signal is approximately equal between LL three and LR three.

Next, make a YFP area versus CFP area by variate plot with quadrants. Adjust the YFP parameter in the compensation matrix until the MFI of the YFP signal is approximately equal between the quadrants. Similar to that shown previously For fret flow cytometry.

The primary fluorescent spillover occurs from CFP emitted into the fret detection channel, which can be corrected via fluorescence compensation using the single color positive control cells When all of the gates have been set and the CFP signal has been compensated. Highlight the wells of interest and run the remainder of the plate to analyze the data. First, define the cells and single cells using the same parameters as we're setting up the flow analysis.

Next, using cell line three, plot the YFP single positive cells into a fret versus YFP by variant plot. Draw a polygonal gate runs along and away from the slope of the fret positive cell population to define a false fret gait, which excludes YFP spillover into the fret filter. After applying the false fret gait to all of the samples, plot the empty liposome treated C-F-P-Y-F-P cells onto a fret versus CFP by variate plot and introduce a polygonal ga along the slope of the population that extends upward and leftward away from the population.

Any events that shift into this gate are considered fret positive and the percentage of cells will correlate to the seed titer used during treatment. Finally, record the analysis parameters of interest, including the percent fret positivity and the median fluorescence intensity of the fret positive cells. Then measure the product of the percent positivity and MFI to manually calculate the integrated fret density.

In the absence of tau aggregates or seeds, tau is maintained in a soluble monomeric state as represented by a consistent diffuse and fret negative fluorescent signal. Following the introduction of tau seeds into the biosensor cells, endogenous tau is converted into an aggregated and fret positive state fret quantification. Following flow cytometry analysis confirms that femto molar concentrations of recombinant tau are sufficient for detection.Here.

Brainstem tissue from tauopathy mice sacrificed at different ages, serves as a representative brain region demonstrating a consistent detection of seeding activity across the animals as early as 1.5 months of age. In this experiment, brain homogenous from Alzheimer's disease subjects exhibited robust seeding activity, whereas age matched control and Huntington's disease subjects did not demonstrate the assay specificity to tau aggregates untreated primary neuronal cultures transduced with tau, CFP, and tau. YFP lentiviral constructs exhibited diffuse fluorescent signal upon treatment with recombinant tau seeds.

However, endogenous tau forms large aggregates detectable by fret flow cytometry even in the absence of liposome mediated transduction. When seed sources are pre incubated with drugs that inhibit tau uptake such as heparin and are treated in the absence of liposomes, the seeding activity contained within both recombinant fis and tauopathy mouse brain samples can be blocked. Once mastered.

This technique can be completed in three days with each day only one to two hours of bench work. When attempting this procedure, it is important to remember that in-house optimization will be required for certain steps. For example, when determining effective titers from protio pathic seeds from various biological sources.

After watching this video, you should have a good understanding of how to set up, run and analyze fret flow cytometry experiments for the purposes of detecting protio pathic seeding activity from recombinant and biological sources.