2.5. Stool Bacterial Extracellular Vesicle Extraction and Removal of Exosomes

Isolation of Vesicles from Stool Samples

Modified by Chanel Mosby from: Wu, J. et al. A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS. J. Proteomics Bioinform. 12, 38–42 (2019).

Materials

• 25 mg of mice stool
• PBS, dPBS
• dPBS + 1X Protease inhibitor cocktail
• Ultra centrifuge tubes for the small swinging rotor
• 3 screw-cap 2 ml tubes
  • Recommended to label as: pellet, S/N, bEV

Procedure

---benchtop ---

1. Weigh out 25 mg of stool into a screw-top 2 ml tube labeled “pellet”. 
2. In the same tube, add 500 ml of PBS and suspend the stool pellets by extensive vortexing. 
   1. It is recommended to use the horizontal tube holder attachment for the vortex as this will take several minutes. You should see the stool dispersed fairly evenly throughout the liquid, however it is normal for fibrous components to still be present. If there are a lot of bubbles forming, reduce the amount of time you are vortexing. 
3. Spin the disrupted pellet in a benchtop centrifuge at 500 x g for 10 minutes to precipitate insoluble materials. Transfer the supernatant (S/N) from the tube into a new sterile 2 ml tube labeled “S/N”. 
4. In the original tube with the stool labeled “pellet”, add 500 ml of PBS to the pellet and vortex again to resuspend. 
5. Repeat steps 3 and 4, three more times so that ~ 2 ml of the S/N is in the “S/N” 2 ml tube. Freeze the remaining pellet in the -80°C freezer. 
6. Spin the 2 ml tube with the S/N for 30 min at 10,000 x g to pellet remaining bacteria. 

---ultracentrifuge ---

1. Filter sterilize the S/N with a .22 um filter into the 4 ml ultracentrifuge tubes. Add dPBS to bring volume up to 4 ml then spin for 2 hr at 4°C at 110,000 g. 
2. Remove the S/N and resuspend in 4 ml PBS then spin for 1 hr at 110,000 x g at 4°C
3. Repeat step 6 two times.
   1. In the end, there should have been one 2-hr spin and three 1-hr spins.
4. After the final spin, remove S/N and resuspend in 500 ul of dPBS + 1X PI cocktail. 
   1. To make cocktail, take a 15 ml conical tube, fill to 10 ml dPBS and dissolve in it 1 tablet of the Pierce Protease Inhibitor in the fridge door.
5. Plate a spot on LB (10 ul) to ensure no bacterial contamination
6. Next day, remove exosomes and then save the flow through to run Nanosight analysis. Save at 4C if need to do TEM, otherwise can freeze until doing microBCA