This protocol reduces the technical complexity and time required for the enrichment of extracellular vesicles from Mycobacterium tuberculosis, Mtb EVs. It provides a gentle alternative to traditional ultracentrifugation and density-based techniques. The main advantage of this technique is that it is easy to perform, making it more reproducible.
It provides a high yield of Mtb EVs with minimal non-EV protein co-enrichment. Demonstrating the procedure will be Joanie Ryan, a former graduate student of our lab. To begin, prepare a 100-kilodalton molecular weight cutoff centrifugal filter by adding the full volume capacity of PBS.
Centrifuge for five minutes at 2, 800 times G at 4 degrees Celsius. Discard the flowthrough and any remaining PBS, and fill the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2, 800 times G at 4 degrees Celsius until the volume has reduced to the minimum volume of the ultrafiltration device.
Repeat as necessary until the entire sample has been reduced sufficiently. Add 1X PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described before and repeat this step five times to ensure complete washing and buffer exchange.
Then, recover the 100-kilodalton concentrated CFP retentate or 100R, according to the ultrafiltration device specifications. Wash the filter with a minimal volume of 1X PBS at least three times, and pull the wash with the 100R to maximize the recovery. Use several dilution of samples in PBS and quantitate the 100R material with a bicinchoninic assay.
Test the sample in triplicate. Allow the size exclusion chromatography, or SEC column, to equilibrate to room temperature. Turn on the AFC using the power switch on the back of the tower unit and press Setup on the main menu touch screen if necessary to adjust the settings for the load cell.
From the setup screen, align the carousel by selecting Carousel followed by Calibrate. Insert the carousel with the 13 small holes facing up into the AFC tower and adjust the carousel by pressing the minus or plus buttons so that the fluid nozzle is directly above the flush position. Remove the caps from the equilibrated SEC column and slide the SEC column into the appropriate column mount.
Carefully install it on the AFC tower. Ensure that the radio frequency identification tag on the column faces the AFC, and check that the connection between the column and the valve is secure. Place the waste outlet tubing in a collection container.
From the setup screen, select Collection Schedule and set the count to 13 and the size to 0.5 milliliters by pressing the minus or plus buttons. Leave the buffer volume setting as the default of 2.7 milliliters and then close the collection schedule window. Select Start Collection from the home screen and confirm the collection parameters by selecting Yes.
Load 13 labeled 1.7 milliliter microcentrifuge tubes with the lids open and pointing toward the carousel center. Advance the AFC by selecting OK, then mount the column reservoir and advance again by pressing OK.Select the option to flush the column by pressing Yes, and add one column volume of 0.2 micrometer filtered PBS to remove any storage buffer. Once the flush is complete, advance the AFC by pressing OK.Place the carousel cover over the AFC and press OK.Use a pipette to remove any excess buffer from the column.
Bring three milligrams of 100R sample to 500 microliters with 1X PBS and add the sample to the top of the column. Prepare one column volume of 0.2 micrometer filtered PBS. Advance the AFC and allow the sample to run into the fret.
Once the sample has fully entered the column, add the PBS prepared earlier to the reservoir. Monitor the run of the AFC while it collects first the void volume and then the specified fractions. After the run completes and the carousel returns to the waste position, remove the cover and the fraction tubes from the carousel.
For 0.5 millimeters fractions collected from three milligrams of 100R Mtb CFP, measure the protein concentration using the micro-BCA with a 1:3 dilution for fractions numbered 1 to 7 of each sample in triplicate. For 0.5 milliliters fractions numbered 5 to 13, use the BCA with no dilution for each sample in triplicate. Add SDS sample buffer to 10 microliters of each fraction and five micrograms of CFP and 100R.
Boil for five minutes at 100 degrees Celsius. Then load on a 4 to 12%Bis-Tris gel with a molecular weight ladder for polyacrylamide gel electrophoresis, or PAGE. Run an SDS-PAGE gel using 1X MES running buffer and a 200-volt current for 35 minutes.
Remove the gel from the cassette and apply a 50-volt current for a minimum of one hour to transfer the resolved proteins to a 0.2 micrometer nitrocellulose membrane. Finally, perform the Western blot analysis to evaluate the proteins. An SDS-PAGE gel stained with silver demonstrates the overall protein profile for each fraction.
The data shows that the later fractions contain more protein and resemble the 100R material. Western blots detecting LAM, LpqH and GroES demonstrate that protein marker changes across the fractions. Transmission electron microscopy, or TEM images, of SEC fractions 1 to 3 pulled prior to fixation are shown here.
The density gradient ultracentrifugation of Mtb EVs was negatively stained for TEM imaging. The nanoparticle tracking analysis, or NTA distribution, of SEC fractions 1 to 3 is shown here. The density gradient ultracentrifugation Mtb EVs were analyzed with nanoparticle tracking analysis, and the size distribution is displayed here.
This protocol has been optimized for EV purification from three milligrams of 100R. If the protein load is varied, the recommended dilution and BCA strategy on resulting fractions may require adjustment. Mtb EVs prepared with this technique can be used for downstream compositional and omics-based studies.
They are also suitable for in vitro and in vivo experiments. This technique provides a facile and efficient way to produce high-quality Mtb EV preparations, paving the way for increased reproducibility in future Mtb EV experiments.
