Microplate

Primary cultures of Bdnf-Luc mouse cortical cells at 13 DIV were treated with 25 mM KCl (for 6 h), a series of compounds (for 6 h), or herbal extracts (6, 24, or 48 h). Primary cultures of Bdnf-Luc mouse cerebellar granule cells at 7 DIV were treated with 25 mM KCl (for 6 h). Luciferase activity of each well was measured using the Steady-Glo Luciferase Assay System (Promega) with a Glo-Max Navigator microplate Luminometer (Promega), according to the manufacturer’s instruction. White adhesive seal was added to the bottom of the microplate (Perkin Elmer, Waltham, MA, USA) to make it opaque before the measurement of luciferase activity.

The fluorogenic oxidation of dihydroethidium (DHE) to ethidium was used as a measure of superoxide anions to determine the NADPH Oxidase activity [29] (link). In a microplate (Perkin-Elmer), freshly prepared hippocampus homogenates were incubated with DHE (10 µmol), salmon testes DNA (0.5 mg/ml, Sigma)in the presence or absence of β-NADPH (0.1 mM, Sigma) as substrate for 30 min at 37°C in a dark chamber. Ethidium-DNA fluorescence was measured at an excitation of 485±40 nm and an emission of 590±35 nm using fluorescence plate reader (Bio-Tek Instruments, Winooski, VT).

After bacteria with pBSK-Km::ZsGreen were cultured in LB with 50 μg/mL kanamycin at 37°C overnight, the bacteria were resuspended in fresh LB to an OD₆₀₀ of 9. Fifty microliters of this bacterial suspension were pipetted onto NGM plates without or with 2% glucose. Then, approximately 50~60 C. elegans were cultured on these plates for 3 days. After moving from the plates to microscope glass slides, the worms were washed with 0.9% NaCl three times. Subsequently, the C. elegans was paralyzed with 200 mM sodium azide for 5~10 min. Images of nematodes were captured using an upright fluorescence microscope Nikon Ni-E (Nikon, Japan). In order to quantify the fluorescence levels in worms, 20 C. elegans in a well of microplate (PerkinElmer, USA) were detected at 505 nm after 492-nm excitation by Varioskan Flash microplate reader (Thermo Fisher Scientific, USA).