In vitro assays measuring protection by proteins such as cystatin C of primary cortical neuronal and smooth muscle cells.

Neuronal cell culture models have been used to demonstrate the protective effects of cystatin C against a variety of insults, including the toxicity induced by oligomeric and fibrillar amyloid β (Aβ). Here, we describe assays quantifying cystatin C protective effects against cytotoxicity induced by nutrient deprivation, oxidative stress, or cytotoxic forms of Aβ. Three methods for the evaluation of either cell death or cell survival are described: measurement of metabolic activity, cell death, and cell division. The cell culture models used are murine primary cortical neurons and murine primary cerebral smooth muscle cells. The effects of exogenously applied cystatin C are studied by comparing the viability of nonstressed control, stressed control, and cystatin C-treated stressed cells. The effect of endogenous level of cystatin C expression is studied by comparing stressed primary cells isolated from brains of cystatin C transgenic, cystatin C knockout, and wild-type mice.