Purification of Staphylococcus aureus Extracellular Vesicles

EVs were purified from S. aureus N305 culture supernatants using a method adapted from (Gurung et al., 2011 (link)). Sub-cultured cells at the end of exponential phase were diluted 1:1,000 in 1 L of fresh BHI medium and were grown until the stationary phase. After the cells were pelleted at 6,000 g for 15 min, the supernatant fraction was filtered through a 0.22 μm vacuum filter (PES) and the filtrate was concentrated around 100-fold using Amicon ultrafiltration system (Millipore) with 100 kDa filter. The resulting filtrate was subjected to ultracentrifugation at 150,000 g for 120 min at 4°C and were applied to a discontinuous sucrose density gradient (8–68%). After centrifugation at 100,000 g for 150 min at 4°C, each fraction of the gradient was collected. The fractions with density around 1.08–1.13 g/cm³ were then recovered by sedimentation at 150,000 g for 120 min and suspended in Tris-Buffered Saline (TBS) (150 mM NaCl; 50 mM Tris-Cl, pH 7.5). Purified EVs were checked for absence of bacterial contamination and stored at −20°C before use. The EVs amount were measured based on protein concentration using the Bradford reagent (Bio-Rad) and visualized by SDS-PAGE. Hereafter, the S. aureus-secreted vesicle dose correspond to the quantity of S. aureus-secreted vesicle proteins.