Note: This protocol is intended for use with the specific products mentioned within it. Substituting different products is not recommended.

The use of the annexin V apoptosis assay protocol is a common method for detecting apoptotic cells. The below protocols are recommended for use with the specific flow cytometry kits mentioned. Please see the Annexin V Staining page for a discussion about general experimental conditions and avoiding false positives, or to review a selection guide for all of our annexin V products.
 

Annexin V flow cytometry protocols

General notes
  • Invitrogen Fixable Viability Dye (FVD) eFluor 450 is not recommended for use with Annexin V Apoptosis Detection Kits.
  • Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments. 
  • Annexin V can only be used as a marker of apoptosis in cells where the plasma membrane is intact. Destroying the integrity of the plasma membrane will allow binding of Annexin V to PS inside the cell.
     

Annexin V staining protocol

Materials

  • 12 x 75 mm round-bottom tubes 
  • 1X PBS 
  • Annexin V Apoptosis Detection kit (any one of the kits listed). Each kit includes an Annexin V conjugate.
        ° eFluor 450 (Cat. Nos. 88-8006-72, 88-8006-74)
        ° FITC (Cat. Nos. 88-8005-72, 88-8005-74)
        ° PerCP-eFluor 710 (Cat. Nos. 88-8008-72, 88-8008-74)
        ° PE (Cat. Nos. 88-8102-72, 88-8102-74)
        ° PE-Cyanine7 (Cat. Nos. 88-8103-72, 88-8103-74)
        ° APC (Cat. Nos. 88-8007-72, 88-8007-74)
  • 10X Binding buffer

Note: PerCP-eFluor 710 (Cat. Nos. 88-8008-72, 88-8008-74) do not include a viability dye. We recommend using this format in combination with a Invitrogen Fixable Viability Dye (FVD) such as FVD eFluor 660 (Cat. Nos. 65-0864-14, 65-0864-18), FVD eFluor 506 (Cat. Nos. 65-0866-14, 65-0866-18), or FVD eFluor 780 (Cat. Nos. 65-0865-14, 65-8065-18).
 

Experimental procedure

  1. Prepare 1X binding buffer by mixing 1 part of 10X binding buffer with 9 parts of distilled water. 
  2. Harvest cells. 
  3. Wash cells once in 1X PBS, then once in 1X binding buffer. 
  4. Resuspend cells in 1X Binding Buffer at 1-5 x 106 cells/mL. 
  5. Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell suspension. 
  6.  Incubate 10-15 minutes at room temperature. Protect from light. 
  7. Add 2 mL 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at room temperature. Discard supernatant. 
  8. Resuspend cells in 200 µL of 1X binding buffer. 
  9. Add 5 μL of Propidium Iodide Staining Solution or 7-AAD Viability Staining Solution and incubate 5-15 minutes on ice or at room temperature.
    • Note: Propidium iodide and 7-AAD must remain in the buffer during acquisition. Do not wash cells after the addition of propidium iodide or 7-AAD.
  10. Analyze by flow cytometry.
    • Note: Cells should be analyzed within 4 hours after the initial incubation period due to adverse effects on the viability of cells left in the presence of propidium iodide or 7-AAD for prolonged periods. Store at 2–8°C and protect from light until ready for analysis. 

Annexin V staining protocol with Fixable Viability Dyes

Materials

  • 12 x 75 mm round-bottom tubes 
  • 1X PBS 
  • Annexin V Apoptosis Detection kit (any one of the kits listed). Each kit includes an Annexin V conjugate.
        ° eFluor 450 (Cat. Nos. 88-8006-72, 88-8006-74)
        ° FITC (Cat. Nos. 88-8005-72, 88-8005-74)
        ° PerCP-eFluor 710 (Cat. Nos. 88-8008-72, 88-8008-74)
        ° PE (Cat. Nos. 88-8102-72, 88-8102-74)
        ° PE-Cyanine7 (Cat. Nos. 88-8103-72, 88-8103-74)
        ° APC (Cat. Nos. 88-8007-72, 88-8007-74)
  • 10X Binding buffer 
  • Flow Cytometry Staining Buffer (Cat. No. 00-4222) 
  • PBS (azide- and serum/protein-free PBS) 
  • FVD eFluor 660 (Cat. Nos. 65-0864-14, 65-0864-18), FVD eFluor 506 (Cat. Nos. 65-0866-14, 65-0866-18) or FVD eFluor 780 (Cat. Nos. 65-0865-14, 65-8065-18)

Note: FVD eFluor 450 is not recommended for use with the Annexin V Apoptosis Detection Kits.
 

Experimental procedure

  1. Prepare 1X binding buffer by mixing 1 part of 10X binding buffer with 9 parts of distilled water. 
  2. Wash cells twice in azide-free and serum/protein-free PBS. 
  3. Resuspend cells at 1-10 x 106 cells/mL in azide-free and serum/protein-free PBS. 
  4. Add 1 µL of FVD per 1 mL of cells and vortex immediately. 
  5. Incubate for 30 minutes at 2-8°C. Protect from light. 
  6. Wash cells twice with Flow Cytometry Staining Buffer or equivalent. 
  7. Wash cells once with 1X Binding Buffer. 
  8. Resuspend cells in 1X Binding Buffer at 1-5 x 106 cells/mL. 
  9. Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell suspension. 
  10. Incubate 10-15 minutes at room temperature. Protect from light. 
  11. Add 2 mL of 1X binding buffer and centrifuge at 400-600 x g for 5 minutes at room temperature. Discard supernatant. 
  12. Resuspend cells in 200 μL of 1X Binding Buffer. 
  13. Analyze by flow cytometry.

Annexin V staining protocol with surface and intracellular staining

Materials

Note: FVD eFluor 450 is not recommended for use with the Annexin V Apoptosis Detection Kits.
 

Experimental procedure

  1. Prepare 1X binding buffer by mixing 1 part of 10X binding buffer with 9 parts of distilled water. 
  2. Stain cell surface antigen(s). Refer to ‘Staining Cell Surface Targets, Protocol A’. 
  3. Wash cells twice in azide-free and serum/protein-free PBS. 
  4. Resuspend cells at 1-10 x 106 cells/mL in azide-free and serum/protein-free PBS. 
  5. Add 1 µL of Fixable Viability Dye per 1 mL of cells and vortex immediately. 
  6. Incubate for 30 minutes at 2-8°C. Protect from light.  
  7. Wash cells twice in Flow Cytometry Staining Buffer or equivalent. 
  8. Wash cells once with 1X binding buffer. 
  9. Resuspend cells in 1X binding buffer at 1-5 x 106 cells/mL. 
  10. Add 5 μL of fluorochrome-conjugated Annexin V to 100 μL of the cell suspension. 
  11. Incubate 10-15 minutes at room temperature. Protect from light. 
  12. Wash cells once with 1X binding buffer. 
  13. Stain intracellular antigen(s). Refer to ‘Staining Intracellular Antigens, Protocol A or B’. 
  14. Analyze by flow cytometry.
     

Resources

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