A Size Exclusion Chromatography Technique to Purify Mycobacterial Extracellular Vesicles

1. Preparation of crude Mtb EV concentrate

NOTE: For detailed procedures on the cultivation of Mtb and preparation of culture filtrate protein (CFP), see References. It is recommended that bacterial culture media is free from growth supplements with EV-containing or proteinaceous components, such as Oleic Albumin Dextrose Catalase (OADC), and detergents such as Tween. It is also recommended that the bacterial culture's quality and harvested CFP be screened to ensure limited cell death and lysis.

1. Prepare a 100 kDa molecular weight cut-off (MWCO) centrifugal filter (see Table of Materials) by adding the full volume capacity of phosphate-buffered saline (1x PBS). Centrifuge for 5 min at 2,800 x g at 4 °C.
   NOTE: If using a filter with no dead stop volume, care must be taken to ensure the sample volume does not reduce below the filter level, resulting in complete filter drying during centrifugation.
2. Discard the flow-through and any remaining PBS before filling the sample chamber with Mtb CFP to its maximum capacity. Centrifuge at 2,800 x g at 4 °C until the volume has reduced to the minimum volume of the ultrafiltration device. Repeat as necessary, adding more CFP to the unit until the entire sample has been reduced sufficiently.
   NOTE: Retain a portion of the 100 kDa flow-through (100F) material for downstream qualification if desired. Store at 4 °C.
3. Add 1x PBS to the filter unit containing the concentrate up to the total device capacity. Reduce the volume as described in 1.2. Repeat this step five times to ensure complete washing and buffer exchange.
4. Recover the 100 kDa concentrated CFP retentate (100R) according to the ultrafiltration device specifications (see Table of Materials). Once the 100R is recovered, wash the filter with a minimal volume of 1x PBS at least three times, and pool the wash with the 100R to maximize the recovery.
5. Quantitate the 100R material with a bicinchoninic assay (BCA) following the manufacturer's instructions (see Table of Materials). To perform the assay, use several dilutions of samples 1:2, 1:5, and 1:10 in PBS. Test the sample in triplicate.
   NOTE: Retain a portion of the 100R material for downstream qualification if desired. Store at 4 °C.

2. Size exclusion chromatography for the enrichment of Mtb EVs from CFP

NOTE: The following procedure is specific for using 3 mg of 100R Mtb CFP with SEC column and automatic fraction collector (AFC, see Table of Materials). It can be adapted for other starting concentrations and column types by following the manufacturer's specifications. Additionally, the users are recommended to read and understand the automatic fraction collector user manual.

1. Allow the SEC column to equilibrate to room temperature.
2. Turn on the AFC using the power switch on the back of the tower unit and adjust the settings for the load cell, if necessary, by pressing SETUP on the main menu touchscreen. The load cell must only be calibrated upon first use, after every software update, and if inconsistency in fraction collection volumes is noticed.
3. From the SETUP screen, align the carousel by selecting CAROUSEL > CALIBRATE. Insert the carousel with the 13 small holes facing up into the AFC tower, and adjust the carousel so that the fluid nozzle is directly above the flush position by pressing the – and/or + buttons.
4. Remove the caps from the equilibrated SEC column. Slide the SEC column into the appropriate column mount and carefully install it on the AFC tower. Ensure the radio frequency identification (RFID) 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.
5. From the SETUP screen, select Collection Schedule and set the count to 13 and the size to 0.5 mL by pressing the – and/or + buttons. Leave the "Buffer Volume" setting as the default of 2.7 mL, and then close the "Collection Schedule" window by pressing X.
6. Select START COLLECTION from the home screen and confirm the collection parameters by selecting YES. Load 13 labeled 1.7 mL microcentrifuge tubes with the lids open and pointing toward the carousel center.
7. 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 µm filtered PBS to remove any storage buffer. Once the flush is complete, advance the AFC by pressing OK.
8. Place the carousel cover over the AFC and press OK. Prepare one column volume of 0.2 µm filtered PBS. Use a pipette to remove any excess buffer from the column.
9. Bring 3 mg of 100R sample as quantified in step 1.5 to 500 µL with 1x PBS. Add the 3 mg sample to the top of the column. Advance the AFC and allow the sample to run into the frit. Once the sample has fully entered the column, add the PBS prepared in step 2.6 to the reservoir.
10. 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 waste position, remove the cover and remove the fraction tubes from the carousel. Store the fractions at 4 °C prior to quantification (step 3) and qualification (step 4).
11. If the column is to be reused, select the option to clean the column by pressing YES; otherwise, press NO. Follow the instructions on the AFC.
    NOTE: Once the column is washed and the storage buffer has run through, it can be removed, capped, and stored at 4 °C.