The primary culture of central nervous system (CNS) neurons is a popular test system for a rapid, quantitative and reliable assessment of the effects of drugs on central neurons. Consequently, studies on the excitotoxicity of NMDA activation and on intracellular calcium handling machineries with respect to ischemic damage to the brain as well as neurodegenerative diseases have been highly productive (Ankarcrona et al., 1995). This created the need to establish a standard method for assessment of neurotoxicity. Several methods are currently being used, including LDH leakage and MTT assays (Mosmann, 1983; Decker and Lohmann-Matthes, 1988). We have used another common method for assessing acute cell death, the dead/live assay (Slepian et al., 1996). It provides a precise time and concentration evaluation of the process of cell death following exposure to a toxic substance, in our case, zeta-inhibitory peptide (ZIP), previously proposed to act as a selective PKM-zeta antagonist (Ling et al., 2002; Pastalkova et al., 2006; Sadeh et al., 2015). In this assay, we load cells with Calcein-AM, which, upon penetration into live neurons, is converted from a non-fluorescent compound into a highly fluorescent green fluorophore. Subsequently, we expose the neurons to different concentrations of ZIP for various durations, in the presence of propidium iodide (PI) which penetrates dead cells, and count red/green fluorescent cells. This method allows us to examine which cells were alive before, and died after exposure to the toxic substance as well as the time course of cell death.
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