Quantification of Rat Brain BDNF Levels

Following manufacturer guidelines, a Rat BDNF Elisa Kit (Thermo Scientific^TM, Waltham, MA, USA) quantified brain-derived neurotrophic factor (BDNF) [71 (link)]. Briefly, 70 μL of standard and 70 μL of the sample were mixed in 96-well plates on a horizontal shaker for 2.5 h at room temperature, away from light. After thoroughly washing the plate with 1× wash buffer, it was allowed to dry upside down on a paper towel. Subsequently, 70 μL of 1× prepared biotinylated detection antibody was added to each well, followed by incubation for 45 min at room temperature with gentle shaking. After 45 min of HRP-conjugated streptavidin incubation, 66.7 μL of TMB substrate solution was added for 30 min, followed by 33.7 μL of stop solution to stop the reaction. A spectrometer (Infinite 200 Pro MPlex, Tecan, Männedorf, Switzerland) measured absorbance at 450 nm and computed BDNF concentration by comparing findings to the standard curve. Results are presented as mean ± SD (%) vs. control cells (0% line).