Free glycerol colorimetric fluorometric assay kit

Aliquots of cell culture were centrifuged to obtain the supernatant, which was stored at 4 °C for further analysis. The glucose concentration was examined using a Glucose Assay Kit (Sigma-Aldrich). A Free Glycerol Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) was used to determine glycerol levels, and a Succinate (Succinic Acid) Colorimetric Assay Kit (BioVision) was used to examine the succinate levels in the culture supernatant.

RG100204 was formulated in tetraethylene glycol (TEG; Sigma-Aldrich, St. Louis, MO, USA) at 5, 10, and 20 mg/mL, respectively. At t = 0, four groups of 10 mice were removed from food, followed by oral gavage at 2.5 mL/kg with the respective RG100204 formulation, or TEG (0 mg/mL group). Mice were kept fasting throughout the experimental period. Blood samples were collected from the tail vein of restrained animals at 90, 180, 270, and 360 min post-dosing. Blood glucose was measured using a handheld glucometer (ACCUCHEK AVIVA, Roche, Basel, Switzerland), and documented manually. Plasma (10 min centrifugation at 3000× g, 4 °C) was prepared within 45 min from blood collected in Li-heparin tubes and stored on ice until processing. Plasma samples were stored frozen at −20 °C before analysis, using the Free Glycerol Colorimetric/Fluorometric Assay Kit (Biovision, Milpitas, CA, USA), as described previously [3 (link)].
For RG100204 plasma concentration analysis, 20–25 µL Li-heparin blood per time point was frozen on dry ice immediately after collection and then stored at −20 °C until LC-MS analysis.

Glycerol release from adipocytes was used as a measure of lipolysis, and was performed using a free glycerol colorimetric/fluorometric assay kit (BioVision Inc., USA) according to the manufacturer’s instructions.