0.22 μm polycarbonate syringe filter

Ceca of mice were harvested, frozen immediately in liquid nitrogen, and stored at −80°C until further processing. One milligram of cecal content was mixed with 10 μl 70% ethanol solution, and then homogenized with appropriate amounts of glass beads (1.0 mm in diameter; Biospec Products) by vortexing at 3,000 rpm for 10 min. Homogenized samples were centrifuged at 14,000 × g for 10 min, and the supernatants were collected and processed for fatty acid derivatization according to the method described previously (44 (link)). The derivatized supernatants were filtered using a 0.22-μm polycarbonate syringe filter (Millipore, St. Charles, MO, USA). Analysis of SCFAs was performed using high-performance liquid chromatography (HPLC) (HITACHI, Tokyo, Japan). SCFAs were separated using a C18 HTec column (NUCLEODUR, Macherey-Nagel, Düren, Germany) with a 40°C column temperature, flow rate at 1 ml/min, and detection wavelength set to 400 nm.

Freshly collected fecal samples were mixed with 70% ethanol solution at a ratio of 1 mg of fecal sample: 10 μL 70% ethanol, and then homogenized with appropriate amounts of glass beads (1.0 mm in diameter; Biospec Products) by vortexing at 3000 rpm for 10 min. Homogenized samples were centrifuged at 14,000× g for 10 min, and the supernatants were collected for fatty acid derivatization, according to a previously described method [53 (link)]. Derivatized supernatants were filtered using a 0.22-μm polycarbonate syringe filter (Millipore, St. Charles, MO, USA). SCFAs were separated and quantified using high-performance liquid chromatography (HITACHI, Tokyo, Japan) on a C18 HTec column (NUCLEODUR, Macherey-Nagel, Düren, Germany), with column temperature 40 °C, flow rate 1 mL/min, and detection wavelength 400 nm.