This protocol provide guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them. The main advantage of this protocol is that every lab concerned with EVs may limit endotoxin contamination by keeping an aseptic procedure and using simple and widely available equipment. Everyone who is trying this technique for the first time should start with new media, reagents, and plasticware which are low-endotoxin, non-pyrogenic labeled.
For this protocol, use endotoxin-free reagents, water, culture media, filtered PBS, ultra low-endotoxin FBS, and ultracentrifuge tubes. To begin, collect the supernatant from previously cultured SW480 and SW620 cell lines into appropriately labeled 15-milliliter tubes. Remove cell debris by centrifuging the supernatant at 500 x g for five minutes at room temperature.
Without disturbing the debris, collect the supernatant into a new labeled tube, and centrifuge at 3, 200 x g for 12 minutes at four degrees Celsius. Carefully transfer 45 milliliters of the supernatant into a vertically-placed sterile 50-milliliter tube, without wetting the edges of the tube. Wrap the tube's cap with a sterile, transparent film, and store it vertically at minus 80 degrees Celsius for subsequent isolation of extracellular vesicles.
Begin isolation by preparing 0.22-micrometer syringe filters, syringes, and tubes containing approximately 90 milliliters of supernatant. Fill a syringe with the supernatant, and fix the filter to the needle adapter. Filter the supernatant through the filter into a 50-milliliter tube.
Pipette seven milliliters of the filtrate into a prepared ultracentrifuge tube, and centrifuge at 100, 000 x g for two hours at four degrees Celsius. Use a sterile Pasteur pipette to discard the supernatant. Pull the pellets into an ultracentrifuge tube using a long filtered pipette tip.
Rinse the extracellular vesicles by adding seven milliliters of filtered endotoxin-free PBS. After ultracentrifugation, discard the supernatant using a sterile Pasteur pipette and resuspend the pellet in 200 microliters of endotoxin-free PBS. Transfer the suspension to a sterile 1.5-milliliter low protein binding test tube.
Then transfer 10 microliters of suspension to a new 1.5-milliliter tube for further analysis. Wrap the cap of the tube, and store it at minus 80 degrees Celsius. Now, into a new tube, dilute one microliter of suspension using filtered PBS at a ratio of 1 to 1000 for nanoparticle tracking analysis.
To perform blotting analysis, first mix 20 micrograms of the samples with a loading buffer. Incubate the samples at 70 degrees Celsius for 10 minutes. Then load 20 micrograms of the sample into each well of a 10 to 14%polyacrylamide gel with SDS, and perform electrophoresis for 45 minutes at 150 volts, with running buffer.
Next, place the gel in a transfer machine with Towbin buffer, and perform a semi-dry transfer of proteins onto a polyvinylidene difluoride membrane at 25 volts for one hour. Block the membrane with 1%BSA by incubating it for one hour on a rocker. Add BSA diluted antibodies, anti-CD9 or anti-Alix, to the membrane, and incubate overnight at four degrees Celsius on a rocker.
After removing the antibodies, wash the membrane thrice with 10 milliliters of TBST for 10 minutes. Now add goat anti-rabbit or anti-mouse secondary antibody, and incubate again on a rocker for one hour at room temperature. Discard the antibody, and wash the membrane as demonstrated previously.
Pour one milliliter of the solution containing substrate and luminol at an equal ratio on the membrane. Immediately place the membrane in an imaging system, and visualize the protein bands on the screen. In the present study, western blot analysis confirmed the presence of two extracellular vesicle markers, CD9 and Alix.
The mean size and concentrations of the vesicles isolated from both SW480 and SW620 were similar. Stimulation of monocyte with different doses of lipopolysaccharide, or LPS, showed the lowest dose of LPS enabling the secretion of interleukin 10 and tumor necrosis factor in monocytes was 50 picogram per milliliter. The chromogenic LAL test indicated that LPS contamination of the extracellular vesicles was around 50 picograms per milliliter for both cell lines.
The most important thing to remember is the use of LPS-free or depleted reagents, and routine LPS measurement at each step of the procedure. Preventing endotoxin contamination is easier than eliminating it. The proposed protocol limits the possibility of false results due to the contamination of EVs with endotoxin, and allows for assessing the EVs activity per cell.
The proposed protocol is useful for researchers working with endotoxin-sensitive cells, like monocytes, dendritic cells, and macrophages.
