Galaxy 16dh centrifuge

We extracted metabolites from the colon tissues based on previous methods [28 (link),29 (link),30 (link),31 (link)]. Briefly, colon tissues were accurately weighed by difference using an analytical balance (Ohaus; VWR, Mississauga, ON, Canada) into a 1.5 mL microcentrifuge tube (Eppendorf, Mississauga, ON, Canada). Tissues were homogenized in 70% ethanol (200 µL 70% ethanol/100 mg tissue) with a disposable tissue grinder (Kontes Pellet Pestle; Fisher Scientific, Ottawa, ON, Canada). The resulting suspension was centrifuged at 3000× g for 3 min (Galaxy 16DH centrifuge, VWR, Mississauga, Ontario, Canada), and the supernatant was decanted and centrifuge-filtered using a 0.2 µm Ultrafree-MC centrifugal filter (Millipore-Sigma, Oakville, ON, Canada) at 3000× g for 3 min (Galaxy 16DH centrifuge). One hundred µL of the filtrate was transferred to an autosampler vial (300 µL polypropylene with pre-slit Teflon-coated caps; Waters Corp., Mississauga, ON, Canada) fitted with a conical bottom spring insert (250 µL glass; Canadian Life Science, Peterborough, ON, Canada) for ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis.

The vitamin C content was determined according to that described by Escobedo-Avellaneda et al. [30 (link)] with some modifications. Vitamin C in the samples was extracted with TCA 6% using a 1:4 sample-solvent ratio and the mixture was centrifuged at 12,500 g/4 °C for 5 min using a Galaxy 16DH centrifuge (VWR International LLC, Radnor, PA, USA). The supernatant (50 µL) was mixed with 25 µL of potassium phosphate buffer 75 mM, pH 7. For the analysis of reduced ascorbate, 50 µL of distilled water was added to the mixture, then it was incubated for 10 min at room temperature (25 °C). Then 335 µL of a mixture of TCA 10%: H₃PO₄ 43%: bipyridyl 4%: FeCl₃ 3%; in a percentage content of 33%, 26.7%, 26.7%, and 13.3%, respectively were added to all assay tubes. After 1 h incubation at 37 °C, 200 μL of the sample was transferred to a Costar clear 96-flat bottom well microplate (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the absorbance was measured at 525 nm (maximum absorption point confirmed by wavelength scan 400 to 650 nm) in a microplate reader (Synergy HT, BioTek Instruments, Inc., Bad Friedrichshall, Germany). A five-point calibration curve (R² = 0.9948) in a 0.1–1.0 mM range of AA was used to obtain vitamin C concentrations as mg AA/100 g sample on a wet basis. Then, three replicates with triplicates analyses were performed (n = 9) [30 (link)].