Neuronal Viability Assay

Neuronal viability was analyzed using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) at 24 h after treatment with LLT extract (1, 10, 25, 50, 100, 200, and 400 µg/mL) followed by stimulation with or without H₂O₂. Briefly, CCK-8 solution (10 µL) was added to each well and incubated for 4 h at 37°C, and then, absorbance was measured using a microplate reader (Epoch, BioteK, Winooski, VT, USA) at 450 nm. Neuronal viability was expressed as a percentage of the blank group which was defined as 100% viability. Neuronal viability was also determined using a live/dead cell imaging kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The staining solution comprised two probes measuring the recognized cytotoxicity and cell viability parameters calcein AM, indicating live cells (green), and BOBO-3 Iodide (EthD-1), indicating dead cells (red). The culture medium was discarded, and each sample was incubated in 100 µL of staining solution for 15 min at 37°C. To quantify neuronal viability, 10 random images per group were captured at 10× magnification using a confocal microscope (Eclipse C2 Plus, Minato, Tokyo, Nikon, Japan). Live and dead cells were manually counted using ImageJ software (1.37v, National Institutes of Health, Bethesda, MD, USA).