Gs 6r centrifuge

Throughout the study plasma/serum was prepared by centrifuging at 3000 rpm for 10 min at 4 °C (using a GS-6R centrifuge; Beckman, Fullerton, CA, USA). The resulting plasma/serum was stored at −20 °C until analyzed at the completion of the study. All samples from each subject were analyzed within the same analytic run. Samples were analyzed in two batches.
Fasting blood lipids were measured in two consecutive blood samples at baseline (day 1, 2) and end of the 6-week intervention (day 43, 44). Postprandial blood lipids were measured at the end of intervention (day 44) at timepoints −15 min, −5 min and +15 min, +30 min, +45 min, +60 min, +90 min, +120 min, +180 min and +240 min after the mixed meal challenge. Serum TC, LDL-C and HDL-C and TG, and plasma glucose concentrations, were measured using commercial enzymatic kits (Roche Diagnostics, Basel, Switzerland) on a Hitachi 902 auto-analyzer (Roche Diagnostics, Indianapolis, IN). Plasma Lp(a) concentrations were measured using a commercial kit (Randox Pty Ltd, Parramatta, NSW, Australia) on a Beckman AU480 auto-analyzer (Beckman, Fullerton, CA, USA). Serum insulin was measured using a commercial ELISA kit (Mercodia AB, Uppsala, Sweden). All blood measurements were carried out at CSIRO. Non-HDL-C (TC minus HDL-C) and remnant C defined as TC minus (HDL-C and LDL-C) were calculated.

OMV isolations were performed by adapting methods previously described [40 ]. B. burgdorferi were grown in autoclaved 10 mL glass culture tubes to a total volume of 200 mL for seven days. OMVs were isolated from a total of 10⁹–10¹⁰ bacteria. Cultures were pooled and centrifuged at 3200 × g for 60 min in a Beckman GS-6R centrifuge. The supernatant was removed, and the pellet was resuspended in fresh BSK-H with 6% rabbit serum. Bacteria were then incubated at 37 °C, 5% CO₂ for 2 h to promote OMV production. Cultures were pooled again, and the bacteria were pelleted by centrifugation at 3200× g for 60 min. The supernatant was removed and double filtered using a 0.22 µm polyethersulfone (PES) filter (Millipore Cat no: SLGP033RS, Burlington, MA, USA). The bacterial pellet was resuspended in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% SDS), the insoluble portion was pelleted, and the remaining supernatant was stored at −80 °C. For ultracentrifugation, polycarbonate tubes (Beckman #355618, Brea, CA, USA) were used with a type 70 Ti rotor (Beckman #337922, Brea, CA, USA). The supernatant was ultracentrifuged using a Beckman Optima LE-80k ultracentrifuge for 1 h at 100,000× g, 4 °C. The pellet was resuspended in PBS, aliquoted, and stored at −80 °C until needed.

Harvested cells, planktonic or biofilm, were subjected to three freeze-thaw cycles in the extraction buffer, centrifuged (3,000 rpm, 10 min, 5°C, 15 ml tubes, Beckman GS-6 R centrifuge) and resuspended in 700 μl lysis buffer containing 8 M urea, 2% (v/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 64.8 mM dithiothreitol (DTT), 2% immobilized pH gradient (IPG) buffer pH 4–7 (Pharmacia Amersham Biotech, Sweden). The suspensions were then ultrasonicated with homogenizing 0.2 mm glass beads for 4 × 5 minutes (5 second pulses and pauses, amplitude 40, Vibra-Cell^TM Ultrasonic Processor, SONICS), with alternate periods of cooling on ice. Intact cells and cell wall fragments were removed by centrifugation at 17,000 × g for 10 min at 4°C and the recovered supernatants (cellular protein extracts) were stored at −20°C until further processing. The protein concentration was determined using the 2-D Quant kit (GE Healthcare Life Sciences).