Materials and Reagents

Annexin V FLUOS staining kit (F. Hofformann-La Roche, catalog number: 11858777001 )
The kit contains ready-to-use Annexin-V-FLUOS solution, propidium iodide solution, and incubation buffer
Trypsin
NaCl
KCl
Na₂HPO₄
KH₂PO₄
PBS buffer (pH 7.4) (see Recipes)

Equipment

Flow cytometer
Centrifuge
T25 culture flask

Procedure

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)
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).
Wash the collected cells twice with PBS and centrifuge (670 x g, 5 min, RT).
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))
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

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

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.