Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

1. Preparation of buffers

1. To prepare the ELISA wash buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 1 g bovine serum albumin (BSA) to 1 L of deionized (DI) water. Add 500 µL polysorbate-20. Adjust the pH to 7.2 using HCl or NaOH.
2. To prepare the blocking buffer, add 3.94 g Tris-base, 8.77 g NaCl, and 10 g BSA. Add 500 µL polysorbate-20 to 1 L of DI water. Adjust the pH to 7.2 using HCl or NaOH.
3. To prepare the elution buffer (PBS), add 8.01 g NaCl, 2.7 g KCl, 1.42 g Na₂HPO₄, and 0.24 g KH₂PO₄ to 1 L DI water. Adjust the pH to 7.4 using HCl or NaOH.
   NOTE: A 10x solution of this buffer can be made and diluted with DI water as needed.

2. Preparation of OMV sample

1. Grow A. actinomycetemcomitans cells to the late exponential phase (optical density at 600 nm of 0.7). Pellet the cells by centrifuging twice at 10,000 x g at 4 °C for 10 min. Filter the supernatant through a 0.45 µm filter.
2. Concentrate the bacteria-free supernatant using 50 kDa-molecular weight cut-off filters. Ultracentrifuge the concentrated solution at 105,000 x g at 4 °C for 30 min.
3. Resuspend the pellet in PBS and ultracentrifuge again (105,000 x g at 4 °C for 30 min.) Resuspend the pellet in 2 mL of PBS.

3. Packing the S-1000 column

1. Mix the stock bottle of gel filtration medium with a glass stir rod and pour out into a glass bottle the volume necessary to fill the column, plus approximately 50% excess (about 135 mL). Let these beads sit until they have settled, and then decant off the excess liquid. Resuspend the beads in elution buffer, so that the final solution is approximately 70% (by volume) gel, 30% buffer. Degas the solution under vacuum.
2. Mount the glass column vertically using a ring stand and fill with elution buffer to wet the walls of the column. Drain the buffer until there is only about 1 cm of buffer remaining in the column.
3. Without creating bubbles, carefully pipette beads into the column, filling the column to the top. Continue to drain excess buffer throughout this process. Be sure to not let the beads settle completely before adding additional beads to the top of the column. The column should be packed to a height of about 2 cm below the bottom of the column reservoir.

4. Loading the sample and collecting fractions

1. Degas the elution buffer under vacuum. Wash the column with two column-volumes (180 mL) of elution buffer.
2. Allow the remaining buffer to fully enter the column. Once the buffer has reached the top of the gel layer, carefully pipette a 2-mL sample containing OMVs (at a lipid concentration of approximately 100 – 200 nmol/ L) onto the surface of the beads, being careful not to disturb any of the beads at the top of the column. Allow the sample to fully enter the gel, that is, when no liquid remains above the gel layer.
3. Carefully and slowly add elution buffer on top of the gel column. Do not disturb the top layer of the gel, as this will cause sample dilution.
4. Place a single 50-mL tube under the column and open the column. Collect the first 20 mL of the eluent. Add additional elution buffer to the top of the column, carefully, as needed to ensure the column is never dry.
5. Place a series of 1.5 mL tubes under the column. Start the column and collect a series of 1-mL samples in each tube. As the samples are being collected, continue to add elution buffer to the top of the column, as necessary. Repeat until 96 fractions have been collected. Stop the column.
   NOTE: The samples should be stored at -20 °C for longterm storage or 4 °C for short-term storage until further analysis.
6. To clean the column, run one column volume (90 mL) of 0.1 M NaOH through the column. Run two column volumes (180 mL) of elution buffer through the column.