Cells were washed twice with 10 ml 5% mannitol (Sigma-Aldrich), the mannitol was carefully and completely removed before 0.9 ml 100% methanol was added onto the cells, firmly rocking the dish so that the methanol covers the cell monolayer
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However, the amount of endogenous antioxidants such as superoxide dismutase and catalase were also observed to be lower in smokers in comparison with fertile non-smokers [19, 90]
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