Evaluation of Hippocampal Neuron Cytotoxicity

The level of cytotoxicity in primary hippocampal neurons was assayed by measuring leakage of LDH using an in vitro toxicology assay kit (Sigma-Aldrich) as previously described.^{54 (link)} In brief, data were calculated by finding the activity of LDH leaked into the medium by damaged cells/total LDH activity in the culture (cells plus medium). The culture media and lysed cells were collected after treatment of neurons as described in the relevant figure legends. The LDH assay mixture was made according to the manufacturer’s protocol and added to each sample. After 20 min incubation, the reaction was terminated by adding 1 N HCl. LDH activity was spectrophotometrically measured with a VICTOR^{3 (link)} multilabel reader (PerkinElmer, Waltham, MA, USA) with absorbance set at 490 nm. Calcein-AM and PI: viable or dead cells were stained with Calcein-AM or PI as previously described.^{8 (link), 14 (link)} After treatment of neurons as described in the corresponding figure legends, 25 nM Calcein-AM or 0.5 μM PI (Invitrogen, Molecular Probes, Carlsbad, CA, USA) was added into the culture medium for 30 min. Images were taken using a Zeiss Axiovert 200 microscope. The number of PI positive neurons, or calcein fluorescence densitometry per cell was analyzed using AxioVision 4.8.