Annexin V-FITC staining for detecting apoptosis

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

Induce apoptosis via the desired method.

Collect cells by centrifugation.

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

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

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.

Incubate at room temperature for 5 min in the dark.

Steps

Induce apoptosis via the desired method.

Collect cells by centrifugation.

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

Gently trypsinize and wash cells once with serum-containing media

• This should be done before incubation with annexin V-FITC (steps 4 and 5).

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

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.

Incubate at room temperature for 5 min in the dark.

Stage 2 - Analyze annexin V-FITC binding

Steps

Analyze annexin V-FITC binding via flow cytometry

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

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

Steps

Place the cell suspension on a glass slide.

• Cover the cells with a glass coverslip.

Invert the coverslip on a glass slide and visualize cells.

• Cells can also be washed with 1X Annexin V binding buffer and fixed in 2% formaldehyde before visualization.

Observe cells under a fluorescence microscope using a dual filter set for FITC and rhodamine.

• Cells with bound annexin V-FITC will show green staining in the plasma membrane.
• Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).