The extracellular vesicle field has lacked genetically tractable invertebrate model to study the protein and RNA signals of extracellular vesicles. This protocol establishes the methods for obtaining abundant, pure extracellular vesicles from C.elegans. This is sufficient for robust RNA-SEQ and proteomic analysis.
When the synchronized culture C.elegans have exhausted the food from their normal growth medium plate, use rubber bands to secure a five micrometer nylon mesh filter cap onto a sterile bottle and start a vacuum. Pour the L1 worms onto the filter and pass an equal volume of medium over the worm aggregates. After vortexing, transfer three 10 microliter volumes of the worm suspension onto a worm cultivation plate lid and count the number of animals in each drop.
Adjust the worm suspension to two animals per microliter of fresh medium and vortex the suspension again. Next, add nine, 10 microliter drops of worms onto a new plate lid and manually count the number of animals in each drop. Then calculate the mean and standard error of the mean as a percentage of each worm population and calculate the total number of animals in each population.
To prepare the worms for secretome generation, wash the worms with three five minute washes in 15 milliliters of sterile M9 medium with 50 micrograms per milliliter of carbenicillin per plate per wash with gentle swirling to dislodge the worms, pooling the washes into a 50 milliliter conical tube. After the last wash, dilute the worms to a one animal per microliter of S-Basel solution supplemented with 2.5 micrograms per milliliter of cholesterol and 50 micromolar carbenicillin concentration. Then add up to 400 milliliters of the worm suspension to a sterile two liter bottom baffled flask.
Place the flask on a circular rotator at 20 degrees Celsius and 100 rotations per minute for 24 hours. For extracellular vesicle harvest, transfer the worms in a 50 milliliter conical tube for centrifugation and decant the supernatant through a 0.22 micrometer vacuum filter to remove any particulate debris. After counting, place a drop of suspended worms onto a bacterial lawn and score the animals as moving, paralyzed, or dead after 15 minutes.
Next, concentrate the filtered supernatant to 700 microliters with a regenerated nitrocellulose 10 kilodalton molecular weight cutoff filter and add 150 microliters of fresh S-Basel solution to the reduction. Filter and vortex the resulting solution and repeat the reduction and filtration two more times as just demonstrated. After the last filtration, centrifuge the supernatant to remove any particles and debris that may have accumulated during handling and add protease inhibitor cocktail plus EDTA as per the manufacturer's instructions.
To prepare the size exclusion column, decant 15 milliliters of suspended agarose resin into an empty gravity flow column cartridge. When the column has drained, add S-Basel solution until the resin bed is packed at a final volume of 10 milliliters. Replace the resin buffer with 40 milliliters of sterile filtered S-Basel solution, taking care not to disturb the resin, and allow gravity to drain the buffer through the column.
Once the top of the resin is no longer submerged, cap the bottom of the cartridge to stop the flow and place a collection tube under the column. To initiate the secretome fractionation, remove the cap and immediately add the concentrated secretome solution drop-wise to the top of the column bed, followed by the addition of one milliliter of S-Basel solution drop-wise. Place a fresh collection tube under the column and slowly fill the upper column reservoir with five milliliters of S-Basel solution.
After collecting the first two milliliters of eluate, quickly change the tubes to collect the next four milliliters. Use a regenerated nitrocellulose 10 kilodalton molecular weight cutoff spin filter to concentrate the extracellular vesicle containing column eluate to 300 microliters and transfer the filter retentate to a low binding microcentrifuge tube. Then wash the filter membrane two times with 100 microliters of S-Basel solution at 20 seconds of vortexing per wash and combine the washes with the original retentate for a final sample volume of 500 microliters.
To prepare the extracellular vesicles for quantification by flow cytometric analysis, add 840 microliters of S-Basel solution and 60 microliters of purified extracellular vesicles to a microcentrifuge tube. Add 300 microliters of the sample into two additional microcentrifuge tubes along with seven microliters of an appropriate potentiometric probe per tube. Add seven microliters of 1%Triton X-100 to the last tube and mix all of the tubes by vortexing.
Place a sonicator tip in the center of the tube of the third sample and pulse the sample 10 times at 20%power and a 30%duty cycle. Next, add 300 microliters of S-Basel solution to two control microcentrifuge tubes and add seven microliters of probe to the dye only tube. Label the second tube Buffer Only.
Then incubate all of the tubes for one hour at room temperature protected from light before their analysis by flow cytometry according to standard protocols. To analyze the data, open the files in the appropriate flow cytometric analysis software. Set a rectangular gate starting at the top of the plot spanning from the small angle light scatter level at one times 10 to the two to one times 10 to the four and bringing the gate down until it contains about 2.5%of the total events.
Name the gate after the probe and copy and paste the gate onto the other two sample and control data plots. Then export the analysis to a spreadsheet. Here, representative results from 10 biological replicates are shown.
The variability between replicates is not significant and the typical standard error of the mean of the population size is a little over 10%The transferring of animals between plates results in about a 10%loss of animals, while about 20%of the animals are lost in the sucrose flotation step. On average, 1, 000 wild-type young adult animals will secrete one microgram of proteins larger than 10 kilodaltons into their environment. When this fraction is further separated by size exclusion chromatography, the total protein elution profile demonstrates a small protein peak between two to six milliliters and a large peak after eight milliliters.
The extracellular vesicles are contained within the first five milliliters of the column eluate. In a direct comparison of the flow cytometry characteristics between C.elegans and human extracellular vesicles, a histogram of C.elegans extracellular vesicle sample events, sorted by small angle light scatter, shows that all of the preparations peak at a mean fluorescence intensity of approximately one times 10 to the third. The potentiometric probe DI-8-ANEPPS, robustly labels C.elegans extracellular vesicles and the abundance of purified extracellular vesicles sorted by the DI-8-ANEPPS expression is not significantly different between C.elegans and cell culture derived preparations.
The samples prepared by this procedure are amenable for all typical downstream extracellular vesicle analysis methods, including RNA-SEQ, flow cytometry, nano-particle tracking analysis, and proteomic analysis. Establishing methods for purifying C.elegans extracellular vesicles allows researchers to use this simple invertebrate model to study the genetic pathways and physiological processes that impact extracellular vesicle cargo composition.
