3 kda mwco spin column

Plasma and caecal acetate levels were enzymatically measured in duplicate with a commercial kit (Acetate Colorimetric Assay Kit, Sigma-Aldrich). The analysis of other caecal SCFAs was performed as follows. Caecal samples were suspended in 5% (w/v) metaphosphate-PBS (49 ml/g sample). Suspensions were subsequently filtered with a 3-kDa MWCO spin column (Pall Corporation) and analysed with a GC-2014 gas chromatography-flame ionisation detector system (Shimadzu) equipped with a glass column packed with 60/80 Shincarbon A coated with Themon-3000 (Shinwa-kakou).

For plasma GLP-1 quantification, blood samples were collected in test tubes containing a dipeptidyl peptidase IV inhibitor (Millipore). Colonic GLP-1 was extracted according to the method described by Cani et al.33 (link). The homogenate was centrifuged at 2000 × g for 20 min at 4 °C, and the supernatant fraction was decanted and diluted 1000-fold in assay buffer. The active GLP-1 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) method (Active GLP-1 ELISA Kit, Shibayagi). Plasma and caecal acetate levels were enzymatically measured in duplicate using a commercial kit (Acetate Colorimetric Assay Kit, Sigma-Aldrich). The analysis of other caecal SCFAs was performed as follows: Caecal samples were suspended in 5% (w/v) metaphosphate-PBS (49 mL/g sample). Suspensions were subsequently filtered using a 3-kDa MWCO spin column (Pall Corporation) and analysed on a gas chromatography-flame ionisation detector system (Shimadzu GC2014, Shimadzu, Kyoto, Japan) equipped with a glass column packed with 60/80 SHINCARBON A coated with Themon-3000 (Shinwa-kakou). Plasma triglyceride (Wako) and insulin (Ultrasensitive Mouse Insulin ELISA, Mercodia) levels were determined using the corresponding commercial kits.