2 nbdg

A glucose uptake cell-based assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used to analyze cellular glucose uptake. Briefly, cells (5 × 10⁴/well) were seeded in 96-well plates and incubated overnight in 100 μl of glucose-free medium, reacted with 200 μg/ml 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose) for 1 h in glucose-free medium and then centrifuged for 5 min at 400×g at room temperature to remove the supernatant. Two hundred microliters of the assay buffer was added to each well, the plate was centrifuged for 5 min at 400×g at room temperature, the supernatant was aspirated, and 100 μl of assay buffer was added to each well. 2-NBDG fluorescence was quantified using the infinite M200 PRO (TECAN, Männedorf, Switzerland) with excitation/emission wavelengths of 485/525 nm.

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.

Glucose uptake assay was performed according to the modified method of Alonso-Castro and Salazar-Olivo [20 (link)]. Mature 3T3-L1 adipocytes, cultured on 24-well plates for fluorescence-based assays, were starved in serum-free medium (MEM containing BSA 0.5%) overnight. Subsequently, the medium was replaced with Krebs Ringer phosphate HEPES (KRPH) buffer containing 0.2% BSA (KRPH/BSA) and incubated for 60 min. The cells were then exposed for 60 min to EDB extract suspended in KRPH/BSA buffer supplemented with 80 μM 2-NBDG (2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxy-d-glucose) (Sigma-Aldrich) used as fluorescent glucose analogue. The control cultures were treated with 100 nM insulin or 10 μM rosiglitazone (Sigma-Aldrich). After incubation, cultures were immediately washed three times with ice-cold PBS. The fluorescence intensity of 2-NBDG was measured at λ_ex = 485 nm and λ_em = 535 nm (Tecan M200 Infinite).