The Spectrum Collection (Microsource) consists of structurally diverse compounds covering a large chemical space with 1600 compounds currently marketed or in clinical trials, 300 natural products and their derivatives, and 500 bioactive compounds not yet in clinical trials. For the primary screen and rescreen, 100 nl of 10 mM compounds (in DMSO) was pintooled into each well on DIV13 using the Biomek FXp robot, resulting in 12.5 μM final concentration of compound and 0.125% final DMSO concentration per well. FCCP (100 nl in DMSO) was pintooled into some wells at a 12.5 μM final concentration as a negative control. The primary screen and rescreen were performed with 4 and 10 replicate plates, respectively. Repurchased compounds and peroxide (Luperox, tert-butyl hydroperoxide) for all other experiments were dissolved in DMSO at the desired concentrations and pintooled similarly, keeping the final 0.125% DMSO concentrations in each well.

Glutamate (monosodium glutamate) was dissolved in 50:50% DMSO/water and pintooled as described above. Glutamate and peroxide stocks were made fresh. FCCP was protected from light and kept at −20°C as a 10 mM stock solution dissolved in DMSO to avoid freeze-thaw cycles. Compound plates for all assays were kept at −80°C and thawed just before use.

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