NEW COLORIMETRIC CYTOTOXICITY ASSAY FOR ANTICANCER-DRUG SCREENING
NEW COLORIMETRIC CYTOTOXICITY ASSAY FOR ANTICANCER-DRUG SCREENING
复制标题
DOI：
10.1093/jnci/82.13.1107
复制
发表时间：
1990-07-04
影响因子：
10.3
通讯作者：
BOYD, MR
中科院分区：
文献类型：
--
作者：
SKEHAN, P;STORENG, R;BOYD, MR
我们已经开发了一种快速，灵敏，廉价的方法来测量细胞蛋白质含量的96孔微量滴定板的贴壁和悬浮培养。该方法适用于普通的实验室目的和非常大规模的应用，例如国家癌症研究所的疾病导向体外抗癌药物发现筛选，该筛选每年需要使用数百万个培养威尔斯孔。用溶解在1%乙酸中的0.4%（wt/vol）磺酰罗丹明B（SR B）将用三氯乙酸固定的培养物染色30分钟。通过用1%乙酸洗涤四次除去未结合的染料，并用10 mM未缓冲的Tris碱[三（羟甲基）氨基甲烷]提取蛋白结合的染料，用于在计算机接口的96孔微量滴定板读数器中测定光密度。SRB检测结果与细胞数量呈线性关系，并与Lowry和Bradford检测在从稀疏亚汇合到多层超汇合的密度范围内测量的细胞蛋白值呈线性关系。564 nm处的信噪比约为1.5，每孔1，000个细胞。SRB测定的灵敏度与几种荧光测定的灵敏度相比是有利的，并且上级Lowry和Bradford测定的灵敏度以及其他20种可见染料的灵敏度。SRB测定提供了一个非破坏性、无限期稳定且肉眼可见的比色终点。它提供了一个敏感的措施，药物诱导的细胞毒性，是有用的定量克隆形成，并非常适合于高容量，自动化药物筛选。SRB在488 nm的激光激发下发出强烈的荧光，并且可以通过静态荧光细胞术在单细胞水平上定量测量。
We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute''s disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.