2.11. Neuron viability assay

Cell viability was determined using the CCK-8 assay kit. Before co-culture, astrocytes were grown in 6-well plates (5 × 10⁶ cells/well) and neurons cultured on the transwell insert of 6-well culture plates (5 × 10⁵ cells/well) independently. Astrocytes were first exposed to OGD for 4 h with or without baicalin or MSO, and finally co-cultured with neurons seeded on the inserts. The solo or co-cultured neurons were subjected to OGD/R (2 h/0.5 h) or incubated in DMEM under 95% air and 5% CO₂ for 2.5 h. Neurons on inserts were then incubated with CCK-8 solution for 1 h at 37 °C. The absorbance at 450 nm was measured using a microplate reader (Thermo Varioskan LUX, MA, USA).

To assess the role of astrocytes on glutamate-induced impairment in neurons, astrocytes were first treated with 5 mmol/L succinate for 2 h or exposed to OGD/R for 6 h/30 min in 6-well plates (5 × 10⁶ cells/well), with or without baicalin or MSO, then washed with PBS prior to co-culture with neurons seeded on the inserts of upper chamber (5 × 10⁵ cells/well). All the co-cultured systems were then treated with 2 mmol/L glutamate for 2 h. The survival of neurons cultured on inserts was measured by CCK-8 method as above.