The overall goal of this procedure is to isolate extracellular vesicles from the medium of in vitro cultured adult Schistosoma japonicum. This method can efficiently isolate EV in in vitro culture medium from adult Schistosoma japonicum. The main advantage of this technique is able to efficiently isolate extracellular vesicle from short-term in vitro culture medium, which can minimize the stress potentially result in non-physiological EV secretion.
Demonstration will be by Juntao Liu, a technician from my lab. Collect the parasites in a 500-milliliter beaker, and wash them thoroughly but gently three times using 200 milliliters of warm PBS. Keep the washes brief, and use gravity sedimentation to separate the parasites.
Next, onto 90-millimeter dishes, plate about 200 worm pairs per dish. In the dish, wash the parasites twice more using gravity sedimentation. For these washes, use warm RPMI medium supplemented with pen-strep.
Replace the second wash with 40 milliliters of the medium without antibiotics. Then, culture the parasites at 37 degrees Celsius under 5%carbon dioxide for two hours. After two hours, transfer the media from the plates to centrifuge tubes, and then pool the eggs and worm debris at 2, 000 g for 30 minutes at four degrees Celsius.
Then, collect and transfer the supernatant to a fresh tube. Next, filter the supernatant through a 0.22-micron syringe filter, and collect the filtered solution into a dialyzing bag with a molecular weight cut-off of 3.5 kilodaltons. Then, dialyze the solution using 8%PEG8000 solution at four degrees Celsius overnight.
This step is important for increasing the yield of extracellular vesicles. The next day, transfer the dialyzed solution to a new tube, and add 0.5 volumes of the total exosome isolation reagent. Mix the solution until it is homogeneous, and incubate at about five degrees Celsius overnight.
The following morning, centrifuge the mixture at 15, 000 g for one hour at about five degrees Celsius. Next, aspirate and discard the supernatant, and resuspend the pellet of EVs in two milliliters of PBS. The pellet will not likely be visible.
For western blot analysis, first determine the protein concentration of the sample, such as with the BCA assay. Then, prepare 10 to 20 micrograms of EVs in 22.5 microliters of RIPA buffer. Next, add 7.5 microliters of 4X loading buffer, and heat the mixture at 95 degrees Celsius for five minutes.
After the heat treatment, separate the extracellular vesicle proteins on a 12%SDS-PAGE gel. After running the gel, the proteins are transferred into PVDF membrane. Probe the membrane with anti-CD63 antibodies at a one to 1, 000 dilution.
Then, treat the membrane with anti-rabbit IgG-HRP at a one to 5, 000 dilution. Now, use an ECL detection reagent to develop the membrane, and follow with analysis on a chemiluminescence imaging system. Start with transferring five micrograms of sample into a polypropylene ultracentrifuge tube, and adjust the volume to 12 milliliters with RPMI-1640 medium.
Then, centrifuge the sample at 110, 000 g for 90 minutes at four degrees Celsius. After the centrifugation, carefully aspirate and discard the supernatant. Then, prepare a 2X extracellular vesicle suspension by adding one milliliter of diluent C to the pellet.
Resuspend the pellet with gentle pipetting. Now, prepare fresh 2X dye solution. Add two microliters of the PKH67 ethanolic dye to one milliliter of diluent C in a microfuge tube, and mix well.
Then, stain the sample by mixing together the two solutions and incubating the mix at room temperature for four minutes. Next, stop the staining by adding four milliliters of FBS and waiting another minute. Then, add RPMI-1640 medium to adjust the volume to 12 milliliters, and spin down the suspension to isolate the stained vesicles.
Remove and discard the supernatant. Then, resuspend the pelleted PKH67-labeled EVs in 12 milliliters of RPMI-1640 medium. Then, repeat the centrifugation, but this time collect the vesicles in one milliliter of PBS.
For the uptake assay, prepare overnight cultures of mammalian cells in six-well plates. The next morning, add five micrograms of PKH67-labeled vesicles to each well, and continue the culture for two to four hours. After the incubation, remove the medium, and wash the cells with PBS two times.
Then, fix the cells for 15 minutes, wash them twice with PBS, and stain them with DAPI before imaging. Schistosomes at the liver stage were collected from rabbits 28 days post-infection. Extracellular vesicles were then collected from the schistosomes worms and analyzed.
Their particle size, as determined by Malvern nanoparticle analysis, ranged between 100 and 400 nanometers, with the bulk of the population near 220 nanometers. Transmission electron microscopy revealed the extracellular vesicles to be round and between 100 and 200 nanometers in diameter. Western blotting analysis indicated that the isolated extracellular vesicles were recognized using CD63 antibodies, which is a typical extracellular vesicle marker.
Fluorescence microscopy of PKH67-labeled extracellular vesicles internalized by the NCTC clone 1469 cells showed that the extracellular vesicles were mainly distributed in the cytoplasm of recipient cells. After watching this video, you should have a good understanding of how to isolate EV in in vitro culture medium from adult Schistosoma japonicum.
