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Annexin V-FITC staining for detecting apoptosis

Procedure for the early detection of apoptosis using annexin V-FITC staining and optional propidium iodide (PI).
Last edited Thu 11 Nov 2021

The exposure of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the cell’s surface is an early event in apoptosis and can be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and, therefore, can be used as a probe for detecting apoptosis.

This is an example protocol for PS exposure detection using Annexin V, based on the protocol provided in Annexin V-FITC Apoptosis Detection Kit (ab14085). Please be aware that when working with a specific kit, you should always use the protocol provided on the datasheet as it has been designed for optimal results with the product.

Reagents: 

  • 1X Annexin V binding buffer
  • Annexin V-FITC
  • Optional: propidium iodide
  • 2% formaldehyde
  • Optional: trypsin (for adherent cells)

Stage 1 - Cell incubation with annexin V-FITC

Steps

1

Induce apoptosis via the desired method.

2

Collect cells by centrifugation.

  • You will need to collect 1–5 x 105 cells, depending on the volume your experiment requires.
3

Resuspend cells in 500 µL of 1X Annexin V binding buffer.

4

Add 5 µL of annexin V-FITC

  • You can also add 5 µL of propidium iodide (PI) at this step, should you want to analyze PI staining later.
5

Incubate at room temperature for 5 min in the dark.

Stage 2 - Analyze annexin V-FITC binding

Steps

1

Analyze annexin V-FITC binding via flow cytometry

  • Ex = 488nm and Em = 350nm using FITC signal detector (usually FL1)
2

If propidium iodide was added, analyze PI staining by the phycoerythrin emission signal detector (usually FL2).