Quantifying Glucose Uptake and Consumption

Glucose uptake was measured using nonradioactive fluorescent glucose 2-NBDG (Sigma, St. Louis, MO, USA), as described previously [45 (link)]. For skeletal muscle experiments, fresh GM from mice after insulin or/and PBMT was incubated with 2-NBDG (50 μM) for 30 min. The specimens were washed and lysed, and 2-NBDG levels in GM were quantified using a microplate fluorimeter (Infinite M200; Tecan, Hillsborough, NC, USA). For cell experiments, IR-L6 myotubes were incubated in KHB containing 50 μM 2-NBDG with or without 8 J/cm² PBMT at 37° C for 30 min. Cells were lysed, then 2-NBDG (Ex/Em, 465/540 nm) was quantified with a microplate fluorimeter. For glucose consumption in DMEM medium, IR-L6 myotubes were serum-starved for 12 hours and irradiated by PBMT. The cells were cultured in fresh free-serum medium in a humidified incubator containing 5% CO₂ at 37° C. After 12 hours, glucose consumption in the DMEM medium was measured using Glucose Oxidase Method (Applygen Technologies Inc., Shanghai, China) following the supplier’s instructions.