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

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.