Escherichia coli MG1655 in LB medium was incubated in a shaking incubator at 220 rpm at 37 °C overnight. Then, bacterial cells were removed by centrifuging at 10,000 × g for 10 min at 4 °C (Sorvall ST16R, Thermo Scientific). The obtained supernatant was filtered by 0.45 μm filters (Vacuum Filter System, Corning) and concentrated by centrifugal filters with a molecular weight cutoff (MWCO) of 100 kDa (Millipore). The concentrated supernatant was filtered again through 0.22 μm pore size filters (Millipore) to remove any remaining debris or bacteria. Then, the concentrated medium was centrifuged at 150,000 × g for 3 h at 4 °C (XPN 100, Beckman). The supernatant was removed and the pellet was resuspended in PBS and stored at -80 °C. The protein concentration of OMVs was determined by Micro BCA Protein Assay Kit.
Vacuum filter system
Manufactured by Corning
Sourced in United States
The Vacuum Filter System is a laboratory equipment designed for efficient filtration processes. It utilizes a vacuum to draw liquid samples through a filter, separating the desired components from the liquid. The system is constructed with durable materials and includes components such as a vacuum pump, filter holder, and collection flask. This product provides a reliable and controlled filtration method for various laboratory applications.
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2 protocols using vacuum filter system
1
Isolation and Purification of Bacterial Outer Membrane Vesicles
2
Sterilization of Human Saliva Samples
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This study was approved by Institutional Review Board (IRB No: 2017-010) of Gangneung-Wonju National University Dental Hospital (Gangneung, Korea). After the explanation of the experiment, saliva was collected from participants after informed written consent was obtained. Stimulated saliva was collected from three males more than 20 years of age, who were not smokers, had no systemic diseases and had no caries. The saliva was centrifuged (27,000×g) at 4.0°C for 30 min and the supernatant was separated. Vacuum filtration was performed using 0.45 and 0.22 μm filters (Vacuum Filter system, Corning, NY, USA) sequentially. The filtered saliva was cultured in blood agar plate at 37.0°C and 5.0% CO 2 for 24 h to confirm sterilization. After sterilization was confirmed, the saliva samples were stored at -20.0°C until use in the experiment.
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