Centrifugal device

GF fish were inoculated with secreted bacterial products at 4 dpf by adding cell free supernatant (CFS) at a final concentration of 500 ng/mL to the water of the sterile flasks. CFS was harvested from a 50 mL overnight culture of the specified bacterial strain. The cultures were centrifuged at 7000 g for 10 min at 4°C. The supernatant was then filtered through a 0.22-µm sterile tube top filter (Corning Inc., Corning, NY); sterile supernatant was concentrated at 4°C for 1 hr at 3000 g with a centrifugal device that has a 10 kDa weight cut off (Pall Life Sciences, Port Washington, NY).
For experiments utilizing proteinase K (Qiagen, Hilden, Germany), the enzyme was added to samples of CFS at a final concentration of 100 μg/mL and allowed to incubate at 55°C for 1 hr before inactivating the enzyme at 90°C for 10 min.

Feces were weighed, homogenized in 10% ethanol, and spun at 14,000g for 5 minutes. Supernatant was sent to UCSD’s Glycoanalytics Core. There, Samples were centrifuged at 10,000g for 5 minutes and spin filtered using prewashed 3K spin filtration unit (Centrifugal device, Pall, Life Sciences, Part No. OD003C34). Flow through was spiked with 1.0μg of Inositol as internal standard and lyophilized. Dried material was reduced using sodium borohydride in presence of 1M ammonium hydroxide, overnight at room temperature. Excess reducing agent was neutralized on ice bath using 30% aqueous acetic acid solution. Samples were then dried by nitrogen flush and co-evaporated with 9:1 methanol: acetic acid mixture (3 times) followed by anhydrous methanol (3 times). Finally, the samples were acetylated using mixture of pyridine and acetic anhydride (1:1 v/v) and alditol acetylate of monosaccharides were analyzed by GCMS (Agilent Technologies, 7820 GC System attached with 5975-MSD). Restek-5ms column (30m x 0.25mm x 0.25μm) was used for profiling of the monosaccharides, Ultrapure He was used as carrier gas in split less mode at a flow rate of 1.2mL/min. Quantification of monosaccharides were done based on the response factors obtained from known amount of standard mixture.