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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:

Stage 1 - Cell incubation with annexin V-FITC

Steps

Induce apoptosis via the desired method.

Collect cells by centrifugation.

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

Add 5 µL of annexin V-FITC

Incubate at room temperature for 5 min in the dark.

Steps

Induce apoptosis via the desired method.

Collect cells by centrifugation.

Gently trypsinize and wash cells once with serum-containing media

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

Add 5 µL of annexin V-FITC

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

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.

Adherent cells should be grown directly on a coverslip

Invert the coverslip on a glass slide and visualize cells.

Cells must be incubated with Annexin V-FITC before fixation since any cell membrane disruption can cause non-specific binding of Annexin V to PS on the inner surface of the cell membrane.

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