Glycogen colorimetric fluorometric assay kit

Intracellular glycogen was assessed using the Glycogen Colorimetric/Fluorometric Assay Kit (BioVision). Samples were homogenized in dH₂O, and they were immediately boiled in vented tubes at 100°C for 5 min to minimize glycogen degradation. Homogenates were then centrifuged at 16,000 g for 10 min, and the supernatant was assayed for glycogen according to the manufacturer's instructions. Total glycogen content was then normalized to total cellular protein concentration.

The levels of glycogen in the triceps muscle tissue were determined using a Glycogen Colorimetric/Fluorometric Assay kit (BioVision, USA). MCP-1 and proinflammatory cytokine (TNF-α, IL-6, and IL-1β) concentrations in the triceps muscle tissue were determined using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA; Thermo Fisher, Waltham, MA, USA). The triceps muscle tissue samples were prepared as described previously [8 (link)]. We determined the glycogen and cytokine levels of the triceps muscle because this muscle is the muscle located in the forelimb, which was used to measure the MGS.

To measure hepatic glycogen content, 10 mg of liver tissues obtained from nonfasted, 2 h and 6 h fasted WT Pygl^−/− and Phkb^−/− mice were homogenized. Hepatic glycogen was determined from the liver lysate using a Glycogen Colorimetric/Fluorometric assay kit (K646-100, BioVision). Hepatic free glucose from liver homogenates was determined using a D-Glucose assay kit from Megazyme (Chicago, IL, USA). To determine glycogen and hepatic free glucose content, Glycogen and D-Glucose standard curves were prepared, and samples were analyzed using the SpectraMax i3x (Molecular Device). Hepatic triglycerides were measured using a colorimetric Triglyceride Quantification Kit (K622-100, Biovision). 100–200 mg of liver tissue was homogenized in RIPA buffer containing protease inhibitor cocktail. Crude extract was deproteinized according to kit instructions. Deproteinized hepatic lysates were used to measure Triglycerides (TG).

Cell viability assay was performed using CCK-8 Kit (Dojindo). Briefly, the cells which were seeded into 96-well plates at a density of 1×10⁴ cells/well, were detected with 10 μL CCK-8, and incubated at 37℃ for 4h. The absorbance was measured at 450 nm. In this study, the proliferation index = the absorbance of experimental group—the absorbance of blank group, was used to evaluate the cell viability of each group. Besides, cell proliferation ability was evaluated with Click-iT® Plus EdU Proliferation Kit (Alexa Fluor® 555, Thermo Fisher Scientific), and the assay was mainly performed according to the manufacturer’s instructions.
To further evaluate cell viability, the extracellular/intracellular LDH, intracellular glycogen and glucose in medium in each group were measured with LDH-Cytotoxicity Colorimetric Assay KitⅡ(BioVision), Glycogen Colorimetric/Fluorometric Assay Kit (BioVision) and Amplex® Red Glucose Assay Kit (BioVision) according to the manufacturer’s instruction respectively.

Glycogen levels in liver tissues were determined using the Glycogen Colorimetric/Fluorometric Assay Kit (Biovision, Mountain View, CA) following the manufacturer’s instructions. Glucose uptake and lactate production are reported as glucose utilization and lactate secretion per cell within the observed period, respectively. To that end, glucose and lactate concentrations were measured in supernatants using the Glucose (HK) Assay Kit (Sigma) and Lactate Assay Kit (Sigma) following the manufacturer’s instructions, respectively. Each experiment was performed in triplicate and repeated at least three times independently.