Annexin V Assay

Detecting apoptosis using annexin V and NucleoCounter® NC-3000™

Ascertain if cells are in early or late-stage apoptosis/necrosis
Fast, automated single cell analysis
Standardized results – even between different users
User-friendly protocol with predefined settings
No calibration required
Clear data presentation with Plot Manager feature
Automated PDF reports
Export data in FCS/ACS formats

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Overview of the Annexin V Assay

Externalization, or translocation, of phosphatidylserine (PS) from the inner to the outer membrane leaflet happens in early apoptosis and precedes other apoptotic events such as loss of membrane integrity and DNA fragmentation.

Annexins are a group of cellular proteins that bind to phospholipids in a calcium-dependent manner. A member of this group, Annexin V, has proven to be a useful tool in detecting apoptotic cells since it has a high affinity for PS, and thus translocates with PS to the outer cell membrane.

Using fluorescently labeled Annexin V, the externalization of PS can easily be detected. Annexin V may also bind to PS on late apoptotic and necrotic cells but as the membrane integrity on these cells has been lost, they can be distinguished from early apoptotic cells using propidium iodide (PI). The PI dye is not membrane-permeable and is used to stain cells and DNA.

Assay principle

Using fluorescence microscopy and image analysis, the NucleoCounter® NC-3000™ advanced image cytometer automatically detects apoptotic cells based on PS externalization. Cells are stained with Hoechst 33342, PI and FITC-labeled Annexin V.

Hoechst 33342 stains the total cell population, while Annexin V stains apoptotic and necrotic cells. Early apoptotic cells exclude PI, while late-stage apoptotic and necrotic cells stain positive for both Annexin V and PI.

Data image from the Annexin V Assay where the analyzed cells are shown in the NucleoView™ software.

Results presented in plot manager

Images of processed data from the Annexin V Assay in the NucleoView™ software as histograms and scatter plots.

The diagram demonstrates the effect of adding camptothecin (CPT) on Jurkat cells. CPT is a commonly used topoisomerase poison, which induces apoptosis, leading to the externalization of PS and enabling the binding of Annexin V. Jurkat cells are an immortalized line of human T lymphocyte cells, commonly used for studying the effectiveness of potential anti-cancer compounds.

Jurkat cells were grown in the absence (upper row) or presence (lower row) of camptothecin (CPT). Cells were stained with Hoechst 33342, Annexin V-Fluorescein Isothiocyanate (FITC) conjugated antibody and propidium iodide (PI) and analyzed using the Annexin V Assay with the NucleoCounter® NC-3000™.

Scatter plots and histograms were obtained in the NucleoView™ NC-3000™ software. Quadrants (B, F) and markers (C, D, G, H) in the displayed plots were used to demarcate the various cell populations. In this example, CPT causes a significant increase of early apoptotic cells (marked as Annexin V positive and PI negative cells in the lower right quadrant, F).