Separation of extracellular vesicles from fluids like condition cell culture media can allow researchers to characterize vesticular cargo and explore their intracellular communication mechanisms. This technique allows for sufficient separation of EVs and extracellular proteins from conditioned cell culture media that is straightforward and user-friendly. To begin place PBS in a 37 degrees celsius water bath.
Observe the control and tunicamycin treated cell culture flasks under a compound microscope with a 10 x and 100 x objective lens. Take notes of any differences between the flasks. Remove the medium from the flask and place it in a 50 milliliter tube.
Then centrifuge the tube at 500 G for five minutes at four degrees celsius and transfer the super natant to a new tube. Centrifuge the tube at 2000 G for 20 minutes at four degrees celsius. Transfer the super natant to a new tube and place the tube on ice.
Take four 15 milliliters three kilodaltons cutoff ultra filtration units and add five milliliters of PBS to each tube. Prime the filters by centrifuging the units at 4, 000 G for 10 minutes at four degrees celsius. After removing PBS from the units, divide supernatant from control and treated conditions into two ultra filtration units each with approximately 12.5 milliliters of media in each.
Centrifuge the tubes at 4, 000 G for one hour 45 minutes at four degrees celsius or until the concentrated media or retentate in each unit is concentrated to 250 microliters or less. Transfer the retentate from both control units into a labeled 1.5 milliliter micro centrifuge tube and from treated units into another tube. Measure the total volume of the retentate in each micro centrifuge tube.
If the volume is less than 500 microliters, add 0.22 micron filtered PBS until the final volume is 500 microliters. Place tubes in a minus 80 degree celsius freezer. Turn on the automatic fraction collector or AFC.
Remove the column from four degrees celsius and allow the column to warm up to room temperature. Remove retentate samples from the minus 80 degrees celsius freezer and place them on ice to thaw, while waiting, set eight labeled 1.5 milliliter tubes in the AFC carousel with lids facing inwards. Insert the column into the AFC with the barcode facing the reader and adjust settings to collect eight fractions of 500 microliters each and buffer volume to default.
Then verify the on-screen instructions to start the collection. After the storage buffer is completely absorbed into the top portion of the column matrix. Begin flushing the column by adding 15 milliliters of PBS to the column.
Do not add PBS too early as it will dilute the storage buffer and prevent adequate flushing. Just before all 15 milliliters of PBS get absorbed by the matrix click okay to stop the flushing and remove any residual PBS from the top of the column matrix with a micro pipette. Do not touch the matrix.
Add 500 microliters of sample to the center of the column and click okay to begin the run. After the sample is fully absorbed into the matrix immediately add eight milliliters of PBS to the column. AFC automatically dispels the void volume into the center of the carousel before collecting all eight fractions of 500 microliters each.
After the run, remove fractions from the carousel and place them on ice. Then remove the void volume from the center of the carousel. Next, add 10 milliliters of PBS to the column to wash any residual sample from the column.
And once the repented sample has been fully flushed add 15 milliliters of PBS to the column. As the final PBS is being absorbed by the matrix add 500 microliters of 0.5 molar sodium hydroxide to the center of the column's matrix. Then as the sodium hydroxide is absorbed, add 30 milliliters of PBS to the column and let it flush through.
Once done with all samples preserve the column for later use by adding 15 milliliters of 0.05%sodium azide to the column. Leave approximately five milliliters of sodium azide on the top of the column matrix. Remove the column from the AFC and attach the top and bottom caps before placing the column at four degrees celsius for storage.
Construct the ultra filtration unit. Obtain two milliliters, three kilodalton cutoff ultra filtration units per sample. Each unit is comprised of three parts, cone, filter and flow through cylinder.
Label each part properly. Add one milliliter PBS to the filter portion and cap with the cone piece. Centrifuge the ultrafiltration unit cone side up at 3, 500 G for 10 minutes at four degrees celsius and remove the filtered PBS from the flow through cylinder.
Flip the ultrafiltration unit upside down and centrifuge for two minutes at 1000 G at four degrees celsius to remove any remaining PBS. Combine four 500 microliters of each fraction and add them to the ultrafiltration unit. Then centrifuge columns coincide up for one hour, 45 minutes at 3, 500 G at four degrees celsius or until the retentate level is at 100 microliters.
Remove the flow through cylinder and discard the flow through. Flip the ultra filtration unit upside down and centrifuge for two minutes at 1000 XG at four degrees celsius. Transfer the retentate in the cone to a 1.5 milliliter tube and make the sample volume to 150 microliters with 0.22 micron filtered PBS before placing the tubes in minus 80 degrees celsius freezer.
A representative western block of individual fractions showed strong expression of CD9, CD63, and CD81. Infractions one to four with little or no expression infractions five to eight. No expression of GM130 or calnexin was observed in any fraction.
Albumin was only present in fractions six to eight. The effectiveness of concentrating the EV and protein fractions was also evaluated. Expression of CD9 and CD63 was observed in the cell lysates.
All three CD proteins were present in EV fractions of control and tunicamycin treated samples but not in the protein fractions. The tumor susceptibility gene 101 was present in cell lysates, but not in EV or protein fractions. The GM130 and calnexin were only observed in the cell lysates.
In the western blot of exosome depleted cell culture media, the EV markers were present in the cell lysates but not in the media samples. Albumin expression was observed in the protein fractions and minimal expression in the cell lysates. TEM observations demonstrated spherical structures in the EV fractions of control and tunicamycin treated samples.
These structures were not observed in either sample type protein fractions. Nanoparticle tracking analysis reveals differences in particle concentrations of control and treated fractions in EV fractions compared to the control, slightly more particles were present in the tunicamycin treatment. However, in the protein fractions, fewer particles were detected relative to EVs.
It's important to have the same sample volume for the control and treated conditions, allowing for easy comparison of EVs from the different treatments. After separating EVs from the condition cell culture media we can characterize the protein, RNA and lipid content of the vesicles with proteomics RNA sequencing and lipidomics.
