Welcome guest, Sign in

Home

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method

Cancer Biology > Cell death > Cell biology assays > Apoptosis
Authors: Imayavaramban Lakshmanan
Imayavaramban LakshmananAffiliation: Biochemistry and Molecular Biology, Nebraska Medical Center, Omaha, USA
Bio-protocol author page: a276
 and Surinder K Batra
Surinder K BatraAffiliation: Biochemistry and Molecular Biology, Nebraska Medical Center, Omaha, USA
For correspondence: sbatra@unmc.edu
Bio-protocol author page: a445
Vol 3, Iss 6, 3/20/2013, 10515 views, 1 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.374

[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, Annexin v, Propidium iodide, Necrotic cells

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.


How to cite: 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; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
4/14/2013 4:10:53 PM  

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.

4/15/2013 3:53:35 PM  

Imayavaramban Lakshmanan (Author)
Biochemistry and Molecular Biology,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.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

How to cite
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook