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After two days of growth in liquid medium containing either sucrose, carboxymethylcellulose (CMC, a soluble form of cellulose), or SigmaCell (SMC, insoluble cellulose powder) as the sole carbon source, spent Teredinibacter turnerae fermentation medium was cleared of cells by centrifugation and used for proteomic analysis.
Briefly, total culture medium was cleared by centrifugation.
Conditioned medium was cleared of cells and debris by centrifugation at 4000 r.p.m. for 10 min.
Briefly, the culture medium was cleared via centrifugation at 4,000 g for 10 min at 4°C.
Culture medium was cleared by centrifugation and concentrated using Ultracel-3K Amicon Ultra centrifugal tubes (Merck Millipore Ltd ,Germany).
Collected medium was cleared by centrifugation, filtered, and used undiluted on target cells for 2 hours prior to collection of cell lysates.
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This was repeated 2 or 3 times, until the medium was clear.
The attraction of the medium is clear.
After a further 24 h, the conditioned medium was collected, cleared by low-speed centrifugation, and filtered through 0.45-mm-pore-size PVDF filters.
After 3 days the medium was collected, cleared of cell debris by centrifugation, concentrated approximately 10-fold through a Centriplus YM-10 column (Millipore, Bedford, MA).
After incubation for 24 h, an aliquot of the medium was collected, cleared by centrifugation at 200 g for 5 min and analysed for insulin using the Mercodia Ultrasenstive Rat Insulin ELISA kit.
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