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Compared to an in vivo experiment, neuronal cell cultures are immediately accessible to observation and manipulation. In this protocol, we describe a technique to evaluate the cytotoxicity of a metal, manganese (Mn2+), on hippocampal neuronal cell cultures. Interestingly, this protocol is easily adaptable to any type of primary culture (e.g., cortical neurons) and any type of toxic compound (e.g., chemical product).This protocol is similar to "Neuron-enriched Cultures (Method 2)" protocol (Gao, 2011).