title: Measurement of Acetylcholine from Cell Lines.
abstract: Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lauet al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Songet al., 2003), with some modifications (Lauet al., 2013; Songet al., 2008; Songet al., 2003; Sekhonet al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA.
authors.forename: Jamie K
authors.initials: JK
authors.lastname: Lau
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA.
authors.forename: Kathleen C
authors.initials: KC
authors.lastname: Brown
authors.affiliation: Department of Pharmacology, Physiology, and Toxicology, Marshall University, Huntington, USA.
authors.forename: Piyali
authors.initials: P
authors.lastname: Dasgupta