2.3. Hydrogen Peroxide (H2O2)-Induced Neuronal Injury and IBC Treatments

The rat primary cortical neurons were plated at different densities on poly-D-lysine/laminin plates: 2 × 10⁴ cells/90 µL in 96-well plates for neuronal viability assessment; 4 × 10⁵ cells/450 µL in 24-well plates for immunocytochemistry (ICC); 2 × 10⁶ cells/1.8 mL in 6-well plates for flow cytometry; 4 × 10⁶ cells/2.7 mL in 60-mm² dishes for real-time PCR. After allowing 2 h for attachment and stabilization, H₂O₂ was added to the cortical neurons to reach a final concentration of 500 µM, and the cells were further incubated for 30 min. After this, the H₂O₂-containing medium was discarded and replaced with a new cortical culture medium containing various concentrations of IBC extract (5, 10, 20 µg/mL). The plates were then incubated in 5% CO₂ at 37 °C for 24 h (Figure 1A shows the schematic diagram illustrating the experimental design). In addition, to assess the synaptic density and longest neurite length by use of a synaptic marker for ICC, the cells were prepared in 24-well plates at a density of 4 × 10⁵ cells/450 µL and incubated for 7 days in vitro (DIV). The 7 DIV cortical neurons were treated and incubated for 24 h as described in Figure 1A.