Esa coulochem 3

BAT samples were dissected on a chilled plate, and immediately flash-frozen in dry ice before being stored at −80 °C until further analysis. The frozen tissue was subsequently homogenized, sonicated, and centrifugated at 14,000 g for 20 min at 4 °C. The resulting supernatant fraction was filtered and then injected into a high-performance liquid chromatography (HPLC) system (Shimadzu LC Prominence; Shimadzu Corporation) [27 ,28 (link)]. To separate norepinephrine (NE), dopamine (DA), and serotonin (5-HT), a reverse-phase analytical column (Waters Symmetry 300C18; Waters) was employed. The mobile phase consisted of a 10% MeOH solution (pH = 4) containing 70 mM KH₂PO₄, 1 mM octanesulfonic acid, and 1 mM EDTA, delivered at a flow rate of 1 ml/min. Detection of the neurotransmitters was achieved using a coulometric electrochemical detector (ESA Coulochem III; Thermo Scientific). The first and second electrodes of the analytical cell were set at +50 mV and +350 mV, respectively, while the guard cell was set at −100 mV. Data acquisition and processing were performed using the Shimadzu LC solution software (Shimadzu Corporation). The concentrations of the neurotransmitters were expressed as pg/mg of wet tissue, as shown [27 ,28 (link)].