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.