2.9. In vitro [14C] 2-deoxy-d-glucose uptake ([14C]-2DG)

This protocol was obtained from Dr. Kyriaki Bakirtzi [58], optimized for similar experimental conditions in cerebellar neurons. Primary hippocampal neuronal cells were transferred to fresh serum-free medium 4 h before each experiment. Plates were washed with glucose-free Krebs-Ringer HEPES (KRH) medium and treated with 100 nM insulin or vehicle control (KRH medium) for 15 min. Treatments (IND or vehicle [KRH medium] control) were then added to the medium for 15 min. A mixture of [¹⁴C]-2DG (PerkinElmer Cat. No. NEC720A050UC specific activity 250–350 mCi/mmol) and unlabeled 2-deoxyglucose was added for 3.5 min at 37 °C. Glucose uptake was stopped by washing plates several times with ice-cold KRH with 25 mM glucose and 10 µM cytochalasin b to stop further glucose uptake. Cells were collected in glucose-free KRH containing 0.1% sodium dodecyl sulfate (SDS), and radioactivity was determined by liquid scintillation counting in a Beckman Scintillation Counter.