This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

Stefania Morrone
Dept. Experimental Medicine & Pathology,
University "La Sapienza",
Rome, Italy


 1. Introduction

2A. Basic protocol

 2A.1. Materials

2A.2. Methodology 1. Wash 2x 1x106 cells with PBS.

2. Dilute FITC-Annexin V at a concentration of 1 mg/ml in binding buffer and resuspend cells in 1 ml of this solution (prepare it freshly each time).

3. Incubate 10 min in the dark at RT.

4. Add to the cell suspension 0.1 ml of PI solution prior to analysis to give a final concentration of 1 mg/ml.

5. Analyze cells by flow cytometry:

- acquire data with CellQuest or LYSYS II or any software for phenotyping analysis;

- collect 10,000 events per sample;

- exclude debris by scatter gating (forward vs. side);

- display data as two-color dot plot with FITC-Annexin V (green fluorescence, X axis) vs. PI (red fluorescence, Y axis).


2B. Support Protocols

2B.1. To detect apoptosis and cell phenotype at the same time, cells can be first incubated with the appropriate predetermined concentration of PE-conjugated mAb, then washed with PBS and incubated with FITC-Annexin V according the basic protocol. The cytofluorimetric analysis is performed with PE (orange) as third fluorescence.

2B.2. Annexin V can also be conjugated to biotin. The secondary detection reagent can be the streptavidin-FITC conjugate. To this purpose follow the basic protocol except for substituting FITC-AnnexinV with biotin-Annexin V, then wash cell with binding buffer; add the secondary reagent and incubate 20 min at RT. Continue with the basic protocol from step 4.

3. Commentary

3.1. Background information

3.2. Critical parameters  3.3. Troubleshooting 3.4. Anticipated results  

3.5. Time considerations


3.6. Key references

1. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A. M., Keehnen, R. M. J., Pals, S. T., and van Oers, M. H. J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415.

2. Homburg, C. H., de Haas, M., von dem Borne, A. E., Verhoeven, A. J., Reutelingsperger, C. P., and Roos, D. 1995. Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 85: 532.

3. Vermes, I., Haanen, C., Steffens-Nakken, H., and Reutelingsperger, C. 1995. A novel assay for apoptosis - flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Meth. 184: 39.

4. Fadok, V. A.,Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148: 2207.


Appendix 1 (A1): Stock solutions
Binding buffer  Hepes buffer: 10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2 


PI solution Propidium iodide 10 mg/ml in binding buffer 

 Appendix 2 (A2): Reagents

Annexin V-FITC


  Appendix 3 (A3): Equipment

FACSCalibur 34012422
Becton Dickinson


Appendix 4 (A4): Glossary

ANNEXINS: a family of proteins, known for the anticoagulant properties, structurally related, with Ca2+-dependent phospholipid-binding capacity.

FITC: fluorescein isothiocyanate; fluorochrome commonly used to label proteins (mAb) for green fluorescence.

PE: phycoerythrin; fluorochrome commonly used to label proteins (mAb) for orange fluorescence.

PROPIDIUM IODIDE (PI): nucleic acid dye; phenanthridinium intercalator, which emits red fluorescence when intercalated in nucleic acid.

PHOSPHATIDYLSERINE (PS): a negatively charged phospholipid present on the inner surface of cell plasma membrane.