Fluoro carbonyl cyanide phenylhydrazone (fccp)

Cells were plated at 5000 cells per well (96-well plate) and incubated overnight at 37 °C, 5% CO₂. Serial dilutions of peptides were added to the cells and incubated for up to 24 h. Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP, Abcam) was included on each plate as a control compound for establishing cell fluorescence when mitochondrial membranes are depolarized. FCCP was added 10 min prior to staining to a final concentration of 50 μM. Mitochondrial membrane staining was achieved by adding tetramethylrhodamine, ethyl ester, perchlorate (TMRE, Invitrogen) to a final concentration of 0.5 μM. Control wells with PBS ± TMRE were included on each plate. The plates were incubated for 30 min at 37 °C, 5% CO₂ prior to removing the media and washing the cells two times with 0.2% BSA in PBS. Fluorescence was measured using a Tecan infinite M1000Pro multiplate reader (λ_ex = 549 nm, λ_em = 575 nm). Relative fluorescence was determined: (sample − PBS_−TMRE)/(PBS_+TMRE − PBS_−TMRE). Replicate plates were prepared where multiple time points were measured, and each plate contained at least three technical replicates for every peptide and control treatment.

Mitochondrial membrane potential (Δψ) was measured using the fluorescent lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM, Invitrogen, Molecular Probes, Carlsbad, CA, USA), which accumulates within mitochondria in a potential-dependent manner.^{54 (link), 55 (link)} After treatment with ABT-737 (Selleck Chemical, Houston, TX, USA), glutamate or a combination of both, primary hippocampal neurons were stained with 5 nM TMRM for 30 min at 37 °C in the dark. Images were taken using a Zeiss LSM 710 confocal scanning microscope and TMRM fluorescence densitometry was analyzed using ZEN software (Carl Zeiss Microscopy GmbH, Jena, Germany). Brain mitochondria: immediately after isolation of mitochondria from rat brain, mitochondria were incubated with 2 mM malate, 2 mM glutamate and 2 mM ADP (Sigma), and then treated as described in the figure legends. Mitochondria were mixed with 100 nM TMRM and loaded onto a 96-well plate (500 μg/well). Each well was treated with CsA (2 μM, Cell Signaling, Danvers, MA, USA), FCCP (100 μM, Abcam, Cambridge, UK), ABT-737 (100 nM or 5 μM), ΔN-Bcl-xL, full-length Bcl-xL or a combination and incubated for 20 min in the dark. The intensity of fluorescence was measured by a SpectraMax Gemini XS (Molecular Devices, Sunnyvale, CA, USA) with excitation 544 nm and emission 590 nm.