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The samples were diluted in 18 mL PBS and purified by ultracentrifugation at 42,000 rpm using a 50.2 Ti rotor (Beckman Coulter) for 2.5 h on a 40% (w/v) sucrose cushion

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Typically around 2 to 4 days after the last cigarette, though this varies depending on how regularly someone smokes and individual factors like metabolism

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