Our research focuses on progressive neurodegenerative diseases characterized by the accumulation of the nuclear protein TDP-43 in the cytoplasm, such as ALS or FTLD-TDP. We aim to understand how TDP-43 pathologies spread through the brain in a prion-like manner. We previously found that the release of TDP-43 via extracellular vesicles is mediated by the dysregulation of autophagy.
Progranulin of the of FTLD-TDP facilitates this process. The next challenge is to clarify how autophagy regulates the production and release of extracellular vesicles containing TDP-43, which could lead to the discovery of therapeutic targets for ALS or FTLD-TDP. To begin, take the cultured HeLa cells, count them using a cell counter, and seed one times ten to the power of six cells on a six-centimeter plate containing a culture medium.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and controlled humidity for 24 hours. Next, prepare a 20-micromolar siRNA solution. Mix 100 microliters of reduced serum medium and three microliters of siRNA in a low-retention tube to prepare Solution A.Then, mix 100 microliters of reduced serum medium and five microliters of transfection reagent in a low-retention tube to prepare Solution B.After that, mix Solution A and Solution B, then incubate the mixture at room temperature for five minutes.
Now add the mixture of Solutions A and B to the medium used for culturing HeLa cells and incubate for 24 hours. On the next day, wash the cells with PBS and expose the cells to 500 microliters of 0.25%trypsin and one millimolar EDTA solution at 37 degrees Celsius with 5%carbon dioxide and controlled humidity for five minutes. Next, add 2.5 milliliters of the culture medium to the cells and using a Pasteur transfer pipette with a 20-microliter pipette tip attached, prepare a single-cell suspension.
After counting the cells, seed one times ten to the power of six cells on a ten-centimeter plate and incubate, as demonstrated earlier. At the end of the incubation, prepare the culture medium containing either vehicle or 100 nanomolar bafilomycin A1.Then expose the cells to the medium containing either vehicle or 100 nanomolar bafilomycin A1 and incubate for 24 hours. The following day, collect all the culture medium from the 10-centimeter plate.
Transfer the collected medium into a 15-milliliter tube and centrifuge it at 3, 000 G for 10 minutes at four degrees Celsius to remove cell debris. To isolate the P1 extracellular vesicle fraction, transfer the supernatant after centrifugation at 3, 000 G into a centrifugation tube. Centrifuge the tube at 20, 000 G at four degrees Celsius for one hour.
For the isolation of the P2 extracellular vesicle fraction, transfer the supernatant after centrifugation at 20, 000 G into an ultra-centrifuge tube. Centrifuge it at 110, 000 G at four degrees Celsius for one hour. After wiping off the excess moisture inside the tube with a laboratory wipe, resuspend the remaining pellets in the centrifugation tube in 50 microliters of two-time sample buffer to prepare the P1 extracellular vesicle fraction.
Then remove the supernatant by decanting. Wipe off the excess moisture inside the tube with a laboratory wipe and resuspend the remaining pellets in the ultracentrifugation tube in 50 microliters of two-time sample buffer to prepare the P2 extracellular vesicle fraction. Incubate the P1 and P2 extracellular vesicle fractions at 100 degrees Celsius on a heat block for 10 minutes.
After transferring the culture medium from the 10-centimeter plate into a 15-milliliter tube, wash the cells in the 10-centimeter dish with PBS. Then expose the cells to two milliliters of 0.25%trypsin and one millimolar EDTA solution at 37 degrees Celsius with 5%carbon dioxide and controlled humidity for five minutes. Add eight milliliters of growth medium to the cells.
Using a Pasteur transfer pipette with a 200-microliter pipette tip attached, pipette the cells to prepare a single-cell suspension. Transfer the cell suspension into a 15-milliliter tube and centrifuge it at 1, 000 G for 10 minutes at four degrees Celsius. Then remove the supernatant using an aspirator.
Add PBS to the cell pellet and centrifuge at 1, 000 G for five minutes at four degrees Celsius. After removing the PBS, sonicate the cell pellet in one milliliter of A68 buffer containing 1%sarcosil. Mix 300 microliters of the cell lysate with 100 microliters of four-time sample buffer.
Incubate the mixture at 100 degrees Celsius on a heat block for 10 minutes to prepare the cell fraction.
