This protocol makes it possible to guarantee a good isolation of extracellular vesicles and notification of a higher number of EBDF proteins. Compared to other isolation techniques, this one maintains that it is more integrity and vertical activities. EVs are being studied more and more in LC as well as pathological conditions.
This method can be applied to any systems that we choose to characterize and study the EVs. For that content or the biological ethics special attention is required when iterating EVs, as in the culture results in the following experiments. A visual demonstration is critical to host specific equipment such as a homemade size exclusion chromatography column as well as the involvement of nanoparticle counter and mass spectrometer.
Helping to demonstrate the procedure will be Antonella Raffo-Romero, a from the laboratory. Start by pre-isolating extracellular vesicles, or EVs, from condition medium. Transfer the condition to culture medium from microglia or macrophage cultures into a conical tube and centrifuge it at 1, 200 times G for 10 minutes to pellet the cells.
Transfer the supernatant into a new conical tube and centrifuge for another 20 minutes to eliminate apoptotic bodies. Then transfer the supernatant into a 10.4 milliliter polycarbonate tube. Place the tube into a 70.1 TI rotor and ultra centrifuge at at 100, 000 times G for 90 minutes at four degrees Celsius to pellet the EVs.
After centrifugation, discard the supernatant and resuspend the pellet in 200 microliters of 0.2 micrometer filtered PBS. To isolate the EVs, prepare a homemade size exclusion chromatography column by washing and sterilizing a glass chromatography column, and placing a 60 microliter filter at the bottom. Stack the column with cross-linked agarose gel filtration base matrix to create a stationary phase of 0.6 centimeters in diameter and 20 centimeters in height.
Then rinse the phase with 50 milliliters of 0.2 micro meter filtered PBS. If necessary, store the column at four degrees Celsius for later use. Place the resuspended EV pellet on top of the stationary phase and collect 20 sequential fractions of 250 microliters while continuing to add PBS to the top of the stationary phase.
The fractions can be stored at minus 20 degrees Celsius. To perform a nanoparticle tracking analysis, make a dilution of each fraction with 0.2 micrometer filtered PBS and vortex it to eliminate aggregates. Put the solution in a one milliliter syringe and place it in the automated syringe pump.
Next, adjust the camera settings to the appropriate screen gain level and camera level and click on Run. Load the sample in the analysis chamber with an infusion rate of 1000 for 15 seconds. Then decrease and stabilize the speed flow for video recording to an infusion rate of 25 for 15 seconds.
Capture a three consecutive 60 second videos of the particle flow. Then adjust the camera level and the detection threshold before video analysis. Click on Settings OK to start the analysis, and click on Export when done.
Wash the system with one milliliter of 0.2 micrometer filtered PBS between each fraction. If performing electron microscopy analysis, use a 50 kilodalton centrifugal filter to concentrate the size exclusion chromatography fractions of interest. For protein extraction, mix 50 microliters of RIPA buffer with the EV sample for five minutes on ice.
Sonicate the samples three times at 500 Watts and 20 kilohertz for five seconds. Then remove vesicular debris by centrifuging at 20, 000 times G for 10 minutes at four degrees Celsius. After isolating the proteins, perform protein migration in the stacking gel of a 12%polyacrylic gel.
Mix the proteins in the gel with Coomassie Blue for 20 minutes at room temperature. Then excise each colored gel piece and cut it into small pieces. Put the gel pieces through a series of successive washes as described in the manuscript.
Then dry them completely with a vacuum concentrator. After drying perform protein reduction with 100 microliters of 100 millimolar ammonium bicarbonate containing 10 millimolar Dithiothreitol for one hour at 56 degrees Celsius. Next perform protein alkylation with 100 microliters of 100 millimolar ammonium bicarbonate containing 50 millimolar iodoacetamide for 45 minutes in the dark.
Wash the gel pieces according to manuscript directions and completely dry them on the vacuum concentrator. Perform protein digestion with 50 microliters of trypsin in 20 millimolar ammonium bicarbonate at 37 degrees Celsius overnight. On the next day, extract the digested proteins from the gel with 50 microliters of 100%ACN for 30 minutes at 37 degrees Celsius and then 15 minutes at room temperature with continuous stirring.
Then extract the proteins twice with 50 microliters of 5%TFA in 20 millimolar ammonium bicarbonate, while stirring continuously for 20 minutes. Add 100 microliters of ACN and continue stirring for 10 minutes. Afterwards, dry the proteins and resuspend them in 20 microliters of 0.1%TFA.
Desalt the sample using a 10 microliter pipette tip with C 18 reverse phase media for desalting and concentrating peptides. Then elute them with ACN and 0.1%formic acid. Completely dry the sample with a vacuum concentrator and resuspend it in 20 microliters of ACN and 0.1%formic acid for liquid chromatography tandem mass spectrometry, or LC-MS.
Load the digested peptides into the instrument and perform a sample analysis. To confirm EV isolation, each SCC fraction was subjected to a nanoparticle tracking analysis. The particle number was significantly higher in fractions five, six, and seven.
These fractions were pooled into one sample 2F-EV positive, and compared with EV negative fractions, 1F-EV negative and 3F-EV negative, using Western blot analysis. The results showed the presence of heat shock protein 90 in the EV positive sample and in the cell lysate control. Electron microscopy of the EV positive sample showed EVs in a size range around 100 nanometers and 400 nanometers.
Proteomics analysis was performed in order to identify contaminant proteins in EV negative samples and characterize the protein contents of EVs. The identified proteins were compared between 1F-EV negative and a 2F-EV positive samples, as well as between 2F-EV positive and a 3F-EV negative samples. Using this non-targeted method, a pool of 536 proteins were submitted to the ExoCarta database and led to the identification of 86 EV associated proteins.
In addition, this pool also allowed for the identifying of protein interactions and their associated biological functions. It was found that the proteins from the EV positive sample played roles in immune and neuroprotective pathways. The effects of microglia-derived EVs were evaluated on neurite outgrowth with PC 12 cells and rat primary neurons.
Significant outgrowth increase was observed under EVs compared to control. Macrophage-derived EVs were evaluated on glioma cell invasion. It was found that EVs impaired the growth and invasion of glioma spheroids.
The most important thing to remember is to really carefully discard the supernatant of the ultracentrifugation step in order to prevent the loss of EVs in this procedure. We favor a large scale and non-targeted approach to focus on the whole protein content and then the vertical effect of EVs. so contents in nucleic acids can be associated using analysis.
With this approach from a primary culture, we are able to discriminate between EV proteins and free proteins that are outside of variety. So the question remains whether this coagulation is artifactual or rather some other real
