Year: 2013
Type: article
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) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et 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., 吸烟是肺癌发展的主要风险因素。据估计，吸烟与世界各地80-90％的肺癌病例有关（见参考文献1和2）。香烟烟雾的上瘾成分是尼古丁。我们发表的数据显示，尼古丁促进人支气管肺泡癌细胞（BAC）中乙酰胆碱（ACh）的产生（Lau等人，2013）。 ACh作为人类BAC中的生长因子。以下方案基于由（Song等人，2003）发表的方案，其具有一些修改（Lau等人，2013; Song等人。，2008; Song ，2003; Sekhon et al。，2003）。重要的一点要记住的是胎牛血清（FBS）含有大量的乙酰胆碱（ACh）。因此，细胞必须在无血清培养基中培养以测量培养上清液中的ACh。将两等份的培养物上清液用于分析。该方案在两种条件下测量细胞上清液中的总胆碱：1）在将乙酰胆碱酯酶（AChE）（其将ACh转化为胆碱（也称为总胆碱样品））处理后，和2）在测量样品。通过从总胆碱减去游离胆碱计算样品中的ACh浓度。
Source: BIO-PROTOCOL
Authors: Jamie Lau, Kathleen Brown, Piyali Dasgupta
Institution: Marshall University
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: bronze
OpenAlex ID: W2512969781