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3 protocols using g lca

1

Inflammatory Bowel Disease Biomarker Analysis

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OEA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); DSS (molecular weight=36,000–50,000 kDa) was obtained from MP Biochemical (Santa Ana, CA). MPA O-glucuronide and naloxone 3-β-D-glucuronide were obtained from Toronto Research Chemicals Inc. (ON, Canada). β-MCA, UDCA, HDCA, G-LCA, G-UDCA, G-DCA, T-β-MCA, T-UDCA, T-HDCA and T-LCA were purchased from Steraloids Inc. (Newport, RI). α-MCA, CA, DCA, CDCA, lithocholic acid (LCA), G-CDCA, G-CA, T-CDCA, T-DCA, T-CA, dehydrocholic acid and propranolol were purchased from Sigma-Aldrich (St Louis, MO). The anti-UGT1A, anti-FXR, anti-FGF15 and anti-CYP7A1 antibodies were purchased from Santa Cruz Biotechnology. The anti-PPARα antibody was from Abcam (Cambridge, UK). The anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from SunShine Biotechnology (Nanjing, China). The secondary antibodies and recombinant human FGF19 was obtained from Bioworld Technology (St Louis Park, MN). Other reagents, unless mentioned, were obtained from Sigma-Aldrich. RNA extracts of colon biopsies from 8 healthy humans and 13 ulcerative colitis and Crohn’s disease patients were previously described51 (link). The ulcerative colitis and Crohn’s disease samples were collected from consented patients and approved by the University of Michigan IRB committee (approval number HUM00042210).
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2

Comprehensive Bile Acid Profiling Protocol

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HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Nepean, Ont., Canada). Ultrapure water was prepared by the Milli-Q Ultrapure water purification system (Millipore, Bedford, MA, USA). AOM was obtained from Sigma-Aldrich (St. Louis, MO, USA), and DSS (MW 36000-50000) was purchased from MP Biomedicals (Santa Ana, CA). Ammonium acetate, formic acid, glacial acetic acid and the other reagents (analytical grade) were purchased from Nanjing Chemical Factory (Nanjing, China). ‘Mouse Total Bile Acids Kit’ was purchased from Crystal Chem Inc (Downers Grove, United States). ‘MiniBEST Bacterial Genomic DNA Extraction Kit’ was from TaKaRa Bio Inc (Dalian, China). Anti-FXR, FGF15 and CYP7A1 antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-GAPDH antibody was from Bioworld Technology (St. Louis Park, MN, USA).
CA, CDCA, DCA, and LCA, as well as their glycine [38 (link)] and taurine [45 (link)] conjugates, G-CA, G-CDCA, T-β-MCA, T-CA, T-CDCA, T-DCA, and internal standard dehydrocholic acid (dhCA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). α-MCA, UDCA, HDCA, G-LCA, G-UDCA, G-DCA, T-β-MCA, T-UDCA, T-HDCA and T-LCA were purchased from Steraloids, Inc. (Newport, Rhode Island, USA).
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3

Comprehensive Bile Acid Quantification

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Deuterated-cholic acid (d4-CA), bile acids (CA, CDCA, LCA, TCA, UDCA, taurine, βMCA, TαMCA, TβMCA, TLCA, TCDCA), and formic acid were purchased from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). All other bile acid standards (GCDCA, GCA, GDCA, GHDCA, GLCA, GUDCA, HCA, HDCA, ILA, TDCA, THDCA, TUDCA) were purchased from Steraloids Inc. (Newport, RI, USA). Acetonitrile (ACN) and methanol (MeOH) of Optima LC-MS grade quality were purchased from Thermo Fisher Scientific (Auckland, New Zealand).
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