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Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method
流式细胞术检测Annexin V确定细胞凋亡   

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参见作者原研究论文

本实验方案简略版
Oncogene
Feb 2012

Abstract

This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

Keywords: Apoptosis (凋亡), Annexin v (膜联蛋白V), Propidium iodide (iodide碘化), Necrotic cells (necrotic细胞)

Materials and Reagents

  1. Annexin V FLUOS staining kit (F. Hofformann-La Roche, catalog number: 11858777001 )
  2. The kit contains ready-to-use Annexin-V-FLUOS solution, propidium iodide solution, and incubation buffer
  3. Trypsin
  4. NaCl
  5. KCl
  6. Na2HPO4
  7. KH2PO4
  8. PBS buffer (pH 7.4) (see Recipes)

Equipment

  1. Flow cytometer
  2. Centrifuge
  3. T25 culture flask

Procedure

  1. Seed cells (1 x 106 cells) in a T25 culture flask (in triplicate for experiments) and three T25 culture flask for control (unstained, Annexin only, and propidium iodide only)
  2. After 48 h incubation, collect the supernatant (floating apoptotic cells) and trypsinize the adherent cells (~2 x 106 cells) from each T25 flask (combine respective floating and trypsinized cells resulting in six tubes).
  3. Wash the collected cells twice with PBS and centrifuge (670 x g, 5 min, RT).
  4. Re-suspend each pellet (~2 x 106 cells) in PBS (400 μl).
    For experimental cells (Triplicate) - (400 μl of cells + 100 μl of incubation buffer with 2 μl of Annexin [1 mg/ml] and 2 μl of propidium iodide [1 mg/ml]).
    For control cells
    Control 1: (Unstained) - (without any stain (400 μl of cells + 100 μl of incubation buffer)
    Control 2: (Annexin V only) - (400 μl of cells + 100 μl of incubation buffer with 2 μl of Annexin (1 mg/ml))
    Control 3: (Propidium iodide only) - (400 μl of cells + 100 μl of incubation buffer with 2 μl of Propidium iodide (1 mg/ml))
  5. Analyze the cells using a flow cytometry without washing the cells
    Cells that were propidium iodide (PI) negative and Annexin V negative are considered healthy, cells, PI negative and Annexin V positive cells are considered apoptotic, and cells that are positive to both PI and Annexin V considered necrotic (Figure 1).


    Figure 1.

Recipes

  1. PBS buffer (pH 7.4)
    8 g NaCl
    0.2 g KCl
    2.3 g Na2HPO4
    0.22 g KH2PO4
    In 800 ml of distilled H2O adjust the pH to 7.4

Acknowledgments

The authors thank Dr. Palanimuthu Ponnusamy Moorthy and Dr. Dhanya Haridas for critical reading of the apoptosis experimental protocol. We also thank Philip Hexley and Victoria Smith, Flow Cytometry Research Facility at UNMC, for their support.

References

  1. Lakshmanan, I., M. P. Ponnusamy., S. Das., S. Chakraborty., D. Haridas., P. Mukhopadhyay., S. M. Lele and S. K. Batra (2012). MUC16 induced rapid G2/M transition via interactions with JAK2 for increased proliferation and anti-apoptosis in breast cancer cells. Oncogene 31(7): 805-817.

简介

该测定用于计数已经历凋亡的细胞的数目。 通过用膜联蛋白V和碘化丙啶溶液初始染色细胞,随后通过流式细胞术分析来检测细胞凋亡。 它是基于这样的原理,正常细胞本质上是疏水的,因为它们在内膜(面向细胞质的一侧)表达磷脂酰丝氨酸,并且当细胞经历细胞凋亡时,内膜翻转成为外膜,从而暴露磷脂酰丝氨酸。 暴露的磷脂酰丝氨酸由膜联蛋白V检测,碘化丙啶染色坏死细胞,其具有有助于区分凋亡和坏死细胞的渗漏DNA含量。

关键字:凋亡, 膜联蛋白V, iodide碘化, necrotic细胞

材料和试剂

  1. 膜联蛋白V FLUOS染色试剂盒(F.Hofformann-La Roche,目录号:11858777001)
  2. 该试剂盒包含即用型Annexin-V-FLUOS溶液,碘化丙啶溶液和培养缓冲液
  3. 胰蛋白酶
  4. NaCl
  5. KCl
  6. Na 2 HPO 4
  7. KH sub 2 PO 4
  8. PBS缓冲液(pH 7.4)(参见配方)

设备

  1. 流式细胞仪
  2. 离心机
  3. T25培养瓶

程序

  1. 在T25培养瓶(对于实验重复三次)和用于对照的三个T25培养瓶(未染色,仅膜联蛋白和仅碘化丙啶)中培养种子细胞(1×10 6个细胞)
  2. 孵育48小时后,收集上清液(浮动凋亡细胞),并胰蛋白酶化来自每个T25烧瓶的贴壁细胞(〜2×10 6个细胞)(结合相应的漂浮和胰蛋白酶化细胞,得到6个管)。
  3. 用PBS洗涤收集的细胞两次,离心(670×g,5分钟,室温)。
  4. 重悬在PBS(400μl)中的每个沉淀(〜2×10 6个细胞)。
    对于实验细胞(一式三份) - (400μl细胞+100μl含有2μl膜联蛋白[1mg/ml]和2μl碘化丙啶[1mg/ml]的温育缓冲液)。
    对于控制单元<​​br /> 对照1 :(未染色) - (无任何染色(400μl细胞+100μl孵育缓冲液)
    对照2 :(仅膜联蛋白V) - (400μl细胞+100μl含有2μl膜联蛋白(1mg/ml)的孵育缓冲液)
    对照3 :(仅碘化丙啶) - (400μl细胞+100μl含有2μl碘化丙啶(1mg/ml)的温育缓冲液)
  5. 使用流式细胞仪分析细胞而不洗涤细胞
    作为碘化丙啶(PI)阴性和膜联蛋白V阴性的细胞被认为是健康的,细胞,PI阴性和膜联蛋白V阳性细胞被认为是凋亡的,并且对PI和膜联蛋白V都是阳性的细胞认为是坏死的(图1)。


    图1.

食谱

  1. PBS缓冲液(pH 7.4)
    8克NaCl
    0.2克KCl
    2.3g Na 2 HPO 4
    0.22g KH 2 PO 4 sub/
    在800ml蒸馏的H 2 O中调节pH至7.4

致谢

作者感谢Palanimuthu Ponnusamy Moorthy博士和Dhanya Haridas博士对凋亡实验方案的批判性阅读。 我们还要感谢菲利普·希斯利和维多利亚·史密斯,UNMC的流式细胞术研究设施,他们的支持。

参考文献

  1. Lakshmanan,I.,M.P.Ponnusamy,S.Das,S.Chakraborty,D.Haridas,P.Mukhopadhyay,S.M.Lele和S.K.Batra(2012)。 MUC16通过与JAK2的相互作用诱导快速G2/M转换,用于增加乳腺癌的增殖和抗凋亡 细胞。 Oncogene 31(7):805-817
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引用:Lakshmanan, I. and Batra, S. K. (2013). Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method. Bio-protocol 3(6): e374. DOI: 10.21769/BioProtoc.374.
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Eray Sahin
Yeditepe University
Hi, I'm using this Annexin-V-FLUOS staining kit and according to the protocol prepared by Roche, 100 ul of mixture(incubation buffer+annexin-V+PI) should be mixed with 1 million of cells. Does this number represent the initial amount of cells that have been seeded or for the ones that would be harvested?
Second question; how will the percentages be affected if I use less amount of staining mixture?

Thank you.
4/14/2013 4:10:53 PM Reply
Imayavaramban Lakshmanan
Nebraska Medical Center

Does this number represent the initial amount of cells that have been seeded or for the ones that would be harvested?

Yes. It represents the initial amount of cells that have been seeded

Second question; how will the percentages be affected if I use less amount of staining mixture?

For us it has worked very well for the mentioned concentration, if you want to use less amount of staining solution, we would recommend seeding less number of cells.

4/15/2013 3:53:35 PM