Glycogen colorimetric assay kit 2

Cells were grown to confluency prior to harvesting. Intracellular levels of glycogen were then detected in 1×10⁶ cells using the colorimetric Glycogen Assay Kit II (Abcam) according to manufacturer's instructions.

The intramuscular glycogen content was determined using a colorimetric method (Glycogen Colorimetric Assay Kit II, BioVision Inc., Milpitas, CA, USA) according to the manufacturer’s protocol. Briefly, skeletal muscle samples were homogenized in double-distilled water; subsequently, tissues were boiled in order to inactivate enzymes and then centrifuged. Appropriate reagents were added to the collected supernatants and, after 30 minutes of incubation at room temperature. the absorbance of glycogen products was measured in a hybrid multi-mode microplate reader (Synergy H1TM, BioTek Instruments, Winooski, VT, USA). Calculated values were based on a standard curve, and glycogen concentration was expressed in micrograms per microliter.

Quantitation of starch was performed using Glycogen Colorimetric Assay kit II (BioVision, Milpitas, CA, USA). For sample preparation, 50 μL of C. merolae culture grown in a flat-plate culture apparatus was collected by centrifugation at 1200g for 5 min, and the precipitated cells were resuspended in 90 % ethanol (v/v), and centrifuged at 10,000g for 2 min. This wash process was repeated twice. The washed cells containing starch were completely lysed with 100 μL of 10 N KOH at 100 °C for 5 min and were neutralized with 26 μL of 43.8 N H₃PO₄. The solubilized starch solution was diluted into 20-fold with the Glycogen Hydrolysis Buffer in the (Glycogen Colorimetric Assay) kit. Subsequent manipulation was performed according to the protocol of the Glycogen Colorimetric Assay kit II. Protein content was measured by the Lowry method.

Groups of 20 fly heads in triplicate were used for metabolic measurements. H₂O₂, lipid peroxidation, and triglyceride and glycogen levels were measured using H₂O₂ colorimetric assay (K265), lipid peroxidation (MDA) colorimetric assay (739), triglyceride quantification colorimetric assay (K622), and glycogen colorimetric assay kit II (K648), respectively, according to the manufacturer’s protocols (BioVision). Protein level was measured in parallel for each sample using Pierce BCA Protein Assay Kit, and metabolite levels were normalized to total protein content. Bar graphs showing mean values and standard error of the means (SEMs) were generated using Graphpad, and p-values were obtained using multiple t tests followed by FDR correction according to Benjamini, Krieger, and Yekutieli.

Biochemical analyses were performed in accordance with the manufacturers’ instructions. The levels of complete blood count were assayed on a Sysmex XN-1000 Haematology Analyser (Kobe, Hyogo, Japan). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine, uric acid, lactic dehydrogenase (LDH), ammonia, and creatine phosphokinase (CPK) levels were assayed on a Beckman Coulter UniCel DxC 800 (Brea, CA, USA). Serum lactate levels were measured using Roche Cobas c 501 (Mannheim, Germany). Muscle glycogen contents were measured using the Glycogen Colorimetric Assay Kit II (BioVision, Milpitas, CA, USA).