Xpn 80 ultracentrifuge

B. burgdorferi outer membrane vesicles (OMVs) and protoplasmic cylinders (PCs) were isolated as described (Skare et al., 1995 (link), Lenhart & Akins, 2010 (link)). Briefly, cells were harvested at room temperature by centrifugation for 20 min at 2844 × g, washed in PBS with 0.1 % bovine serum albumin (BSA), and repelleted. The pellet was then resuspended in 38 ml ice-cold 25 mM citrate buffer (pH 3.2) with 0.1% BSA and incubated at room temperature for 2 hours with agitation and a 1-min vortexing step every 30 min. Next, samples were pelleted by centrifugation at 20,000 × g for 30 min and resuspended in 6 ml ice-cold 25 mM citrate buffer (pH 3.2) with 0.1% BSA. This sample was then layered on a discontinuous 56% (wt/wt in 4 ml), 42% (wt/wt in 15.5 ml), and 25% (wt/wt in 12.5ml) sucrose gradient in 25 mM citrate buffer in 38 ml Ultraclear tubes (Beckman-Coulter, 344058). The gradient was centrifuged at 100,000 × g for 18 h at 4°C (Beckman-Coulter XPN-80 Ultracentrifuge, SW32 Ti swinging-bucket rotor) to separate the OMV (upper band) and PC (lower band) fractions. The PC fraction was collected, diluted with PBS, repelleted at 20,000 × g for 20 min, and resuspended in 1 ml PBS with 1mM PMSF for storage at −20°C. The OMV fraction was collected, diluted with PBS, repelleted at 100,000 × g for 4 h at 4°C, and resuspended in 100 μl PBS for storage at −20°C.