Search

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

Download PDF How to cite Favorites 1 Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
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, 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.
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
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.
Q&A

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 including images for the troubleshooting.

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.

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