搜索

[Bio101] MTT Assay of Cell Numbers after Drug/Toxin Treatment
[Bio101] MTT法测定药物/毒物处理后的细胞数量

下载 PDF 引用 收藏 1 提问与回复 分享您的反馈

本文章节

Abstract

MTT assay is a colorimetric method for measuring the activity of enzymes in living cells that reduce MTT to formazan dyes, giving a purple color. It is commonly used to determine cytotoxicity of potential medicinal agents and toxic materials, since these types of materials are expected to stimulate or inhibit cell viability and growth. Here, a general protocol is described to carry out an MTT assay on different types of cells.

Keywords: MTT assay(MTT比色法), Cell numbers(细胞数), Drug(药物)

Materials and Reagents

  1. Raw264.7, MCF-7 or Hela cells
  2. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M5655 )
  3. DMSO
  4. DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250 )
  5. General chemicals (Sigma-Aldrich)

Equipment

  1. 96 well plate
  2. Shaking table
  3. Paper towels
  4. Incubator

Procedure

  1. Plate 500-10,000 cells in 200 μl media per well in a 96 well plate. Leave 8 wells empty for blank controls.
  2. Incubate (37 °C, 5% CO2) overnight to allow the cells to attach to the wells.
  3. Add 2 μl of drug of interest dissolved in DMSO to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the samples into the media.
  4. Incubate (37 °C, 5% CO2) for 1-5 days to allow the drug/toxin to take effect.
  5. Make 2 ml or more of MTT solution per 96 well plate at 5 mg/ml in DPBS. Do not make a stock as MTT in solution is not stable long-term.
  6. Add 20 μl MTT solution to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the MTT into the media.
  7. Incubate (37 °C, 5% CO2) for 1-5 h to allow the MTT to be metabolized.
  8. Dump off the media (dry plate on paper towels to remove residue if necessary).
  9. Resuspend formazan (MTT metabolic product) in 200 μl DMSO. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the formazan into the solvent.
  10. Read optical density at 560 nm and subtract background at 670 nm. Optical density should be directly correlated with cell quantity.

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].

References

  1. Hayon, T., Dvilansky, A., Shpilberg, O. and Nathan, I. (2003). Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma 44(11): 1957-1962.

简介

MTT测定法是用于测量活细胞中酶的活性的比色法,其将MTT还原为甲an染料,得到紫色。 它通常用于确定潜在药剂和毒性材料的细胞毒性,因为这些类型的材料预期刺激或抑制细胞活力和生长。 这里,描述了对不同类型的细胞进行MTT测定的一般方案。

关键字:MTT比色法, 细胞数, 药物

材料和试剂

  1. Raw264.7,MCF-7或Hela细胞
  2. 噻唑基蓝四唑溴化物(MTT)(Sigma-Aldrich,目录号:M5655)
  3. DMSO
  4. DPBS(Life Technologies,Invitrogen TM,目录号:14190-250)
  5. 一般化学品(Sigma-Aldrich)

设备

  1. 96孔板
  2. 摇床
  3. 纸毛巾
  4. 孵化器

程序

  1. 将500-10,000个细胞以200μl培养基/孔接种在96孔板中。 对于空白对照,留下8个空穴
  2. 孵育(37℃,5%CO 2)过夜,以允许细胞附着到孔。
  3. 向每个孔中加入溶于DMSO中的2μl感兴趣的药物。 放置在振动台上,150rpm 5分钟,以将样品充分混合到培养基中。
  4. 孵育(37℃,5%CO 2)1〜5天以使药物/毒素生效。
  5. 使每个96孔板中2ml或更多的MTT溶液在DPBS中为5mg/ml。 不要做股票,因为MTT在解决方案中长期不稳定。
  6. 每孔加入20μlMTT溶液。 放在振动台上,150 rpm 5分钟,以彻底混合MTT到介质中。
  7. 孵育(37℃,5%CO 2)1-5小时,以允许MTT代谢。
  8. 将介质倒出(如有必要,用纸巾上的干燥板清除残留物)。
  9. 重悬甲(MTT代谢产物)在200微升DMSO。 放在振动台上,150 rpm,5分钟,以彻底混合甲into到溶剂中
  10. 读取560nm处的光密度,并在670nm处减去背景。 光密度应与细胞数量直接相关。

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 该方案是在美国加利福尼亚州斯坦福大学遗传学系的Cohen实验室开发的[Chen等人(未公开)]。

参考文献

  1. Hayon,T.,Dvilansky,A.,Shpilberg,O。和Nathan,I。(2003)。 评估基于MTT的测定法作为预测白血病中药物化学敏感性的有用工具。 Leuk Lymphoma 44(11):1957-1962。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Chen, R. (2011). MTT Assay of Cell Numbers after Drug/Toxin Treatment. Bio-protocol Bio101: e51. DOI: 10.21769/BioProtoc.51;
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Yuanqing Lin
How many number of cells should be placed in each well for effective assay?
7/20/2011 5:14:19 PM Reply
bio-protocol

It depends on the cell size, growth rate and treatment time. Usually we let the cells reach 50%-80% confluence before the treatment if the treatment time is within 1 day.

8/31/2011 4:03:58 PM