搜索

Propidium Iodide Staining of Cells for FACS Analysis
流式细胞术检测细胞增殖与凋亡(碘化丙啶法)   

下载 PDF 引用 收藏 2 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA staining can be used to study the cell cycle. Relative DNA content shows the proportion of cells in G1, G2 and S phases. Apoptotic cells show characteristic smear on DNA staining. Here a protocol to stain cells by PI is described.

Keywords: Propidium iodide(碘化丙啶), FACS(流式细胞仪), Cell cycle(细胞周期)

Materials and Reagents

  1. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  2. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
  3. RNase A (Sigma-Aldrich, catalog number: R4642 )
  4. Phosphate buffered saline (PBS)
  5. 70% EtOH
  6. 10 % Triton X-100 (see Recipes)
  7. PI stock solution (see Recipes)

Equipment

  1. Standard table-top centrifuges
  2. FACS machine
  3. Incubator

Procedure

  1. Trypsinize and harvest cells, fix cells into 0.5 ml 70% EtOH (pre-cooled to -20 °C overnight).
  2. Store fixed cells on ice at least 1 h and for up to several days.
  3. Spin down cells for 2 min at 4,000 rpm.
  4. Resuspend cell pellet in 0.5 ml PBS containing 0.25% Triton X-100 and incubate on ice for 15 min.
  5. Spin down the cells for 2 min at 4,000 rpm.
  6. Discard supernatant and resuspend cell pellet in 0.5 ml PBS containing 10 μg/ml RNase A and 20 μg/ml PI stock solution, transfer to FACS tubes and incubate at room temperature (RT) in the dark for 30 min.
  7. Ready for FACS.

Recipes

  1. 10% Triton X-100
    1 ml Triton X-100
    9 ml ddH2O
  2. 1 mg/ml PI stock solution
    10 mg PI
    10 ml ddH2O

Acknowledgments

This protocol was developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

  1. Seki, A., Coppinger, J. A., Jang, C. Y., Yates, J. R. and Fang, G. (2008). Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320(5883): 1655-1658.
  2. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.

简介

荧光活化细胞分选(FACS)用于研究DNA细胞含量。 碘化丙锭(PI)插入双链核酸并发荧光。 它被活细胞排除,但可以穿透死亡或死亡细胞的细胞膜。 因此,PI染色包括在免疫荧光染色方案中以鉴定死细胞。 DNA染色可用于研究细胞周期。 相对DNA含量显示G1,G2和S期细胞的比例。 凋亡细胞在DNA染色上显示特征性涂片。 这里描述了通过PI染色细胞的方案。

关键字:碘化丙啶, 流式细胞仪, 细胞周期

材料和试剂

  1. Triton X-100(Sigma-Aldrich,目录号:T9284)
  2. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4170)
  3. RNA酶A(Sigma-Aldrich,目录号:R4642)
  4. 磷酸盐缓冲盐水(PBS)
  5. 70%EtOH
  6. 10%Triton X-100(参见配方)
  7. PI储备溶液(见配方)

设备

  1. 标准台式离心机
  2. FACS机器
  3. 孵化器

程序

  1. 胰蛋白酶化和收获细胞,将细胞固定在0.5ml 70%EtOH(预冷却至-20℃过夜)。
  2. 将固定细胞在冰上储存至少1小时,最多几天
  3. 以4,000rpm速度旋转2分钟。
  4. 重悬细胞沉淀在0.5毫升含有0.25%Triton X-100的PBS中,并在冰上孵育15分钟
  5. 在4,000rpm下旋转细胞2分钟。
  6. 弃去上清液并将细胞沉淀重悬在含有10μg/ml RNA酶A和20μg/ml PI储备溶液的0.5ml PBS中,转移到FACS管中,并在室温(RT)下在黑暗中孵育30分钟。 >
  7. 准备好FACS。

食谱

  1. 10%Triton X-100 1ml Triton X-100 9ml ddH 2 O 2 /
  2. 1 mg/ml PI储液
    10 mg PI
    10ml ddH 2 O 2 /

致谢

这个协议是在国防部的实验室(斯坦福大学生物系,斯坦福,加利福尼亚州,美国)开发的。 这项工作得到了生物医学研究(G.F.)的Burroughs-Wellcome职业奖和国立卫生研究院(GM062852至G.F.)的资助。

参考文献

  1. Seki,A.,Coppinger,J.A.,Jang,C.Y.,Yates,J.R.and Fang,G。(2008)。 Bora和激酶Aurora协同激活激酶Plk1和 控制有丝分裂入侵。 科学 320(5883):1655-1658
  2. Zhu,H.,Coppinger,J.A.,Jang,C.Y.,Yates,J.R.,3rdand Fang,G。(2008)。 FAM29A通过募集NEDD1-γ-微管蛋白复合物到有丝分裂纺锤体来促进微管扩增。 a> J Cell Biol 183(5):835-848
  • 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。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, H. (2012). Propidium Iodide Staining of Cells for FACS Analysis. Bio-protocol 2(11): e195. DOI: 10.21769/BioProtoc.195.
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。

I found some ref paper where they dis not use Triton X-100.
2/25/2013 5:46:34 PM Reply
Is it possible to do this experiment without Triton X-100?
2/25/2013 5:45:55 PM Reply
Hui Zhu
Stanford University

Triton X-100 is used as permeabilization buffer to penetrate cells. If you don’t penetrate cells, Propidium Iodide won’t stain well.

2/27/2013 1:55:48 PM


Roberta Ruela
Unicamp

You can also use a hypotonic buffer instead of Triton X-100 to permeabilize the cells.

9/4/2013 4:35:06 AM