Optimization and validation of serum deprivation in PC12 cells using 0.1% serum and 50 × 103 cells/well. (a) PC12 cells (passage 12). Values are represented as mean ± SD. Multiple t test, Holm-Sidak method, alpha = 5.000%, **p < 0.01, ****p < 0.0001. (b) PC12 cells (passage 13). Fluorescence measured after 24 h. Values are represented as mean ± SD. Unpaired two-tailed t test, 95% CI, ****p < 0.0001. (c) PC12 cells (passage 9–13). Flupirtine at 3 μM concentration rescued the cells from apoptosis. Values are represented as mean ± SD. Unpaired two-tailed t test, 95% CI, ****p < 0.0001 in comparison to vehicle. ####p < 0.0001 in comparison to serum deprivation. (Inset) Early passage (passage 9–13) and late passage (passage 17–19) PC12 cells grown in 0.1% serum media. LDH release measured after 60 h. Twelve replicates per experiment. Values are represented as mean ± SD. Unpaired two-tailed t test, 95% CI, ****p < 0.0001.