Steritop filtration unit

In order to obtain a sufficient quantity of OMVs to detect a metabolomic signal, 2 l of RM (three culture flasks grown independently) and DM (three culture flasks grown independently) were inoculated with overnight culture of B. thetaiotaomicron at an initial OD of 0.0005 and 0.05, respectively. The OMV extraction was performed as previously described (Stentz et al., 2014 (link)) with slight modifications. After 16 h (OD approximately 4.0), the cell cultures were rapidly cooled in a manually shaken ice bath. The cultures were then centrifuged at 5000 × g for 15 min at 4°C, and the supernatants filtered through a 0.22-μm Steritop filtration unit (Millipore, Billerica, MA, United States) to remove debris and cells. The sterility of the filtrate containing the vesicles was confirmed by plating onto BHI–hemin agar. OMVs in the 2 l filtrates were concentrated by molecular weight (100 kDa MWCO, Vivaflow 200, Sartorius) down to 2 ml, diluted by addition of 1 l of ice-cold PBS, pH 7.4, and the suspensions were filtered and concentrated again, down to 9 ml. The 9 ml retentate was ultracentrifuged [150,000 × g for 2 h at 4°C in a Ti70 rotor (Beckman Instruments)]. The supernatant was completely removed using a vacuum pump and a needle and the OMV pellets were snap frozen in liquid nitrogen and stored at -80°C before extraction.

Bacterial cultures (20 mL) were centrifuged at 5000 g for 15 min at 4°C and the supernatants were filtered through 0.22 μm pore-size polyethersulfone (PES) membranes (Sartorius, Goettingen, Germany) to remove debris and cells. Supernatants were concentrated by ultrafiltration (100 kDa molecular weight cut-off, Vivaspin 20, Sartorius) to 200 μL. The retentate was rinsed twice with 20 mL of PBS (pH 7.4) and concentrated to 200 μL (2 mg dry weight vesicles). OMV sterility was examined by checking for growth of any contaminating bacterial cells on BHI–haemin agar. OMV protein content was determined using the Total Protein Micro protein assay reagent kit (Sigma-Aldrich) after disruption by sonication. Alternatively, the dry weight of OMVs was determined after incubating OMV suspensions in a drying oven for 48 h. OMVs were also collected from the lower compartment of a Steritop filtration unit (Millipore, Billerica, USA) containing sterile BHI–haemin medium on a magnetic stir plate, after their diffusion through a 0.22 μm pore membrane from the upper compartment containing a growing B. thetaiotaomicron culture in BHI–haemin. The sterility of the BHI–haemin containing the vesicles was confirmed by plating OMV suspensions onto BHI–haemin agar immediately before collection.