Colorimetric glucose go assay kit

Primary hepatocytes were isolated by collagenase digestion from mice that had been fasted for 24 h as previously described^{32 (link)}. Cells were washed with PBS and cultured in glucose production buffer consisting of glucose-free DMEM (Ph 7.4), without phenol red, supplemented with 20 mM sodium lactate and 2 mM sodium pyruvate. After incubation with shizukaol F or DMSO, the medium was collected and the glucose concentration was measured with a colorimetric glucose (GO) assay kit (Sigma). The results were normalized to the total protein content.

Cellular glucose consumption was calculated from the difference in glucose levels in medium incubated with and without cells for 24 h. As medium, M199 without phenol red containing 2% FCS and 2 mM glutamine was used and either added to cells or cell-free dishes. If the effect of DCA was studied, the incubation was performed in the presence and absence of 2–20 mM DCA. After 24 h, the medium was removed from dishes and stored at −20 °C. Cells were trypsinized and counted for the normalization of glucose values. To measure glucose, the colorimetric Glucose (GO) Assay Kit (Sigma, Taufkirchen, Germany) was applied according to the manufacturer’s instructions. In this test, glucose oxidation is coupled to the formation of a colored product, oxidized o-dianisidine, whose intensity at 540 nm is proportional to the glucose concentration in the medium. The assay mix contained a 100 µL diluted sample (1:20), or glucose standard and 200 µL assay reagent, including o-dianisidine and glucose oxidase/peroxidase. The reaction was performed in 1.5 mL Eppendorf tubes for 30 min at 37 °C and under gentle shaking and stopped with 200 µL 12 N H₂SO₄. Samples were then transferred to 96-well plates (100 µL per well, triplicates). The analysis was performed using a SunriseTM microplate reader (Tecan GmbH, Crailsheim, Germany) and the Magellan 6 software.