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[Bio101] Cell Cycle Analysis (PI) with GFP Detection
[Bio101] 用GFP检测细胞周期分析(PI)   

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Abstract

Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.

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

Materials and Reagents

  1. Phosphate buffered saline (PBS)
  2. Glucose (Sigma-Aldrich, catalog number: G8270 )
  3. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15170 )
  4. 70% EtOH
  5. Hepes (Sigma-Aldrich, catalog number: H3375 )
  6. NP-40 (MBL International, catalog number: JM-2111-100 )
  7. BSA (Sigma-Aldrich, catalog number: A3803 )
  8. RNase A (Sigma-Aldrich, catalog number: R4642 )
  9. Fix solution (see Recipes)
  10. Wash solution (see Recipes)

Equipment

  1. Centrifuges (Beckman Falcon, TLS-55 )
  2. 15 ml polypropylene falcon tubes (BD Biosciences, Falcon®, catalog number: 352097 )
  3. FACS machine

Procedure

  1. Trypsinize and harvest cells, suspend in 10 ml PBS into 15 ml polypropylene falcon tubes.
  2. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
  3. Wash cells 1x in 5 ml PBS.
  4. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
  5. Thoroughly resuspend cells 1 ml fix solution.
  6. Incubate 10 min on ice.
  7. Add PBS up to 14-15 ml.
  8. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
  9. Wash cells 1x in 5 ml PBS.
  10. Resuspend cells in 0.5 ml 1x PBS. Vortex tube gently and add 4.5 ml ice cold 70% EtOH dropwise over 30 sec to 1 min. Incubate on ice > 1 h.
  11. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
  12. Wash cells 1x in wash solution.
  13. Pellet cells at 1,500 rpm, 3 min, aspirate supernatant.
  14. Resuspend cells in 500 μl of PBS containing 10 μg ml-1 RNase A and 20 μg ml-1 PI, transfer to FACS tubes and incubate at room temperature in the dark for 30 min.
  15. FACS immediately or store at 4 °C until FACS analysis.

Recipes

  1. Fix solution
    1x PBS
    2% Glucose
    3% Paraformaldehyde
  2. Wash solution
    1x PBS
    20 mM Hepes
    0.25% NP-40
    0.1% BSA

Acknowledgments

This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.

References

  1. 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

用GFP构建体感染哺乳动物细胞以过表达或敲低靶基因是研究和操作基因表达的最常用的方法之一。 为了确定细胞周期上的靶基因功能,需要分析GFP阳性细胞与GFP阴性细胞的细胞周期(碘化丙锭,PI染色)。 通常用70%EtOH的细胞的简单固定用于PI染色倾向于淬灭GFP信号; 多聚甲醛(PFA)固定在ETOH固定之前可能有助于维持GFP信号。

关键字:细胞周期, GFP, 流式细胞仪, 碘化丙啶

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)
  2. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  3. 多聚甲醛(Electron Microscopy Sciences,目录号:15170)
  4. 70%EtOH
  5. Hepes(Sigma-Aldrich,目录号:H3375)
  6. NP-40(MBL International,目录号:JM-2111-100)
  7. BSA(Sigma-Aldrich,目录号:A3803)
  8. RNA酶A(Sigma-Aldrich,目录号:R4642)
  9. 修复解决方案(参见配方)
  10. 洗涤溶液(见配方)

设备

  1. 离心机(Beckman Falcon,TLS-55)
  2. 15ml聚丙烯falcon管(BD Biosciences,目录号:352097)
  3. FACS机器

程序

  1. 胰蛋白酶消化和收获细胞,悬浮在10ml PBS中15ml聚丙烯falcon管
  2. 沉淀细胞以1,500rpm,3分钟,吸出上清液
  3. 在5 ml PBS中洗涤细胞1次。
  4. 沉淀细胞以1,500rpm,3分钟,吸出上清液
  5. 彻底重悬细胞1毫升固定溶液
  6. 在冰上孵育10分钟。
  7. 加入PBS至14-15 ml。
  8. 沉淀细胞以1,500rpm,3分钟,吸出上清液
  9. 在5 ml PBS中洗涤细胞1次。
  10. 重悬细胞在0.5毫升1×PBS。 涡旋管轻轻地并且在30秒至1分钟内滴加4.5ml冰冷的70%EtOH。 在冰上孵育> 1小时。
  11. 沉淀细胞以1,500rpm,3分钟,吸出上清液
  12. 在洗涤溶液中洗涤细胞1次。
  13. 沉淀细胞以1,500rpm,3分钟,吸出上清液
  14. 将细胞重悬在500μl含有10μg/ml的RNase A和20μg/ml的PI的PBS中,转移到FACS管中,并在室温下在黑暗中孵育 30分钟。
  15. FACS立即或在4°C储存,直到FACS分析

食谱

  1. 修正解决方案
    1x PBS
    2%葡萄糖
    3%多聚甲醛
  2. 洗液
    1x PBS
    20 mM Hepes
    0.25%NP-40
    0.1%BSA

致谢

这项工作由加利福尼亚再生医学研究所,批准RL1-00100支持。

参考文献

  1. 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。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, H. (2012). Cell Cycle Analysis (PI) with GFP Detection. Bio-protocol Bio101: e199. DOI: 10.21769/BioProtoc.199;
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Wu Sian
06-2757575
Can I use GFP-antibody to improve green florescence and then use PI stain to detect cell cycle? Any protocol can I follow?
9/7/2017 5:55:43 PM Reply
Hui Zhu
Stanford University

I have used GFP antibody in Immunofluorescence staining. For FACS, I have never tried. If GFP+ % is fairly high, you don't need to add antibody. If GFP+ % is very low, adding antibody may not help.

9/7/2017 7:07:44 PM


I have problems to detect the florescence of PI and GFP together, as their emission is very similar. Any suggestions?
Thanks.
6/8/2012 7:06:16 PM Reply
hui zhu
stanford universtiy

PI is much more broader which fluoresces everywhere from yellow to far-red.
You need to do compensation when go with GFP. You can use 7-AAD in place of
PI, which is more tighter.

6/10/2012 2:39:51 PM


Why glucose here?
2/14/2012 12:21:55 PM Reply
hui zhu
stanford universtiy

Paraformaldehyde is godd to penetrate cells. The disadvantage is that paraformaldehyde Only partially destroys osmotic properties of membranes. Osmolarity must be carefully adjusted by glucose or sucrose.

2/22/2012 2:48:32 AM