Efficient and modified 2-NBDG assay to measure glucose uptake in cultured myotubes

Under type-2 diabetes, insulin resistance develops in skeletal muscles as a key defect and to study the disorder, its manifestation, and possible solution, measurement of glucose uptake is a fundamental necessity. Of various approaches (i.e. scintillation counting, flow cytometry, fluorometry and spectrophotometry) fluorescent labelled glucose analogue, 2-NBDG solution is the most popular one. Although 2-NBDG based assay is the most widely used approach in various cells including skeletal muscle, even then all available protocols possess huge variability which impacts the overall data reproducibility. Moreover, starvation (use of glucose/serum free medium), one of the prerequisite condition for glucose uptake assay, itself induces stress specifically during longer pre-incubation periods and alters muscle cell metabolism and morphology, but the fact has not been duly considered. Therefore in the present article, using specific skeletal muscle cells i.e. C2C12 myotubes, we have re-established the conditions like pre-incubation time period, concentrations of insulin, glucose and serum/BSA while maintaining the cultured myotubes in morphologically healthy state. Our lab standardized protocols were observed to be effective in studying insulin resistance condition induced by diverse stresses (oxidative & inflammation) in myotubes. Comparative study conducted with already established protocols demonstrates that the present method is more efficient, effective and better improvised for studying glucose uptake in C2C12.