Cytotox 96 ldh assay

SDS-PAGE western blots and Aβ sandwich ELISAs were performed following standard protocols as previously described [3 (link)]. Primary antibodies used for western blot analysis include: nestin, sox-2, musashi-1 (Millipore), NEP/CD10 (Vector Biolabs, Burlingame, CA, USA), and brain-derived neurotrophic factor (BDNF, Santa Cruz Biotechnology). To test the effects of Aβ treatment on NSC viability cells were treated with 10 nM Aβ42 for 24 hours and then lactate dehydrogenase (LDH) release into the media was measured using the CytoTox 96 LDH assay (Promega Corporation, Madison, WI, USA) following the manufacturer’s protocol.

LDH release was assessed by CytoTox 96^® LDH assay (Promega, Southampton, UK) as previously described [69 (link)], as per manufacturer’s protocol. Briefly, the cell supernatant was mixed with supplied assay buffer and absorbance readings were obtained at 490 nm wavelength using the Varioskan™ Flash microplate reader (Thermo Scientific, Loughborough, UK). The LDH release was presented as fold change compared to control absorbance.

To investigate whether NIV induced cell death via necrosis, apoptosis or a reduction in metabolism, two distinct cell viability assays were performed. Total lactate dehydrogenase (LDH) - reflecting cell number and LDH levels in the culture media (reflecting cell death by necrosis) were determined (CytoTox 96 LDH assay, Promega, UK) according to the manufacturer's protocol. Cell viability was calculated as previously described [13] (link). The 3–(4, 5-dimethylthiazol-2-yl)–2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate whether NIV affected cell metabolism [14] (link).