BestProtocols: Annexin V Staining Protocol for Flow Cytometry

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 staining protocol
• Annexin V staining protocol with Fixable Viability Dyes
• Annexin V staining protocol with surface and intracellular staining
   

• 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.
   

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. 

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.

Materials

• 12 x 75 mm round-bottom tubes 
• 1X PBS (azide- and serum/protein-free PBS) 
• Flow Cytometry Staining Buffer Set (Cat. No. 00-4222)
• Foxp3/Transcription Factor Staining Buffer Set (Cat. No. 00-5523) or Intracellular Fixation & Permeabilization Buffer Set (Cat. No. 88-8824) 
• 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
• 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. 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.