Annexin V-FITC staining protocol for apoptosis detection
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Procedure for the early detection of apoptosis using annexin V-FITC staining and optional propidium iodide (PI).
Reviewed November 11, 2021
Download or print this protocol.
Introduction
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.
Contents
- Reagents
- Cell incubation with Annexin V-FITC
- Analyze Annexin V-FITC binding with flow cytometry
- Observe cell staining with fluorescence microscopy
Reagents
- 1X Annexin V binding buffer
- Annexin V-FITC
- Optional: propidium iodide
- 2% formaldehyde
- Optional: trypsin (for adherent cells)
Method
1. Cell incubation with Annexin V-FITC
1.1 Induce apoptosis via the desired method.
1.2 Collect 1–5 x 105 cells by centrifugation.
1.3 Resuspend cells in 500 µL of 1X Annexin V binding buffer.
1.4 Add 5 µL of annexin V-FITC and 5 µL of propidium iodide (PI, optional).
1.5 Incubate at room temperature for 5 min in the dark.
Alternative: For adherent cells, gently trypsinize and wash cells once with serum-containing media before incubation with annexin V-FITC (Steps 1.3-1.5).
1.6 Proceed to B or C below depending on the analysis method.
2. Analyze Annexin V-FITC binding with flow cytometry
2.1 Analyze annexin V-FITC binding via flow cytometry (Ex = 488 nm; Em = 350 nm) using FITC signal detector (usually FL1)
2.2 If propidium iodide was added, analyze PI staining by the phycoerythrin emission signal detector (usually FL2).
3. Observe cell staining with fluorescence microscopy
3.1 Place the cell suspension from Stage 1.5 on a glass slide. Cover the cells with a glass coverslip.
Alternative: For analyzing adherent cells, grow cells directly on a coverslip.
3.2 Following incubation (Stage 1.5) invert the coverslip on a glass slide and visualize cells. The cells can also be washed with 1X Annexin V binding buffer and fixed in 2% formaldehyde before visualization.
Tip: 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.
3.3 Observe the cells under a fluorescence microscope using a dual filter set for FITC and rhodamine:
- Cells that have 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).