Annexin V Staining

The selection guide below gives an overview of the stand-alone annexin V conjugates available as well as the required binding buffer.

Annexin V stains can be used to evaluate potential apoptosis-inducing drug treatments in live cell. Live cells treated with apoptosis-inducing drugs (i.e. camptothecin) will demonstrate an increase in apoptotic cells (Figure 2, bottom right). 

The selection guide below gives an overview of the assay kits that contain annexin V conjugates. Learn more about Annexin V assay kits below.

During early stages of apoptosis, PS located on the exterior of cells will stain with Annexin V. The intact plasma membrane prevents other dyes such as propidium iodide (PI), 7-AAD, or fixable viability dyes from entering the cell. During late stages of apoptosis, the cell membrane loses integrity and allows annexin V to stain interior PS. The use of additional viability dyes can differentiate between late-stage apoptotic cells and necrotic cells (Figure 3, 4). 

Externalized phosphatidylserine in apoptotic cells is detected using recombinant annexin V conjugated to Pacific Blue (violet-fluorescence) while dead cells are detected using SYTOX AADvanced. Populations can be distinguished by using a flow cytometer with a 405 nm and 488 nm laser lines. A dual parameter plot will show apoptotic cells as violet fluorescence, dead/necrotic cells will be shown as dual red and violet fluorescence, and live cells will be show little to no fluorescence (Figure 5).

Annexin V assays are available to distinguish live (C12 resazurin - Dodecylresorufin), apoptotic (Annexin V), and late apoptotic (SYTOX Green nucleic acid stain) cells (Figure 6). Nonfluorescent C12-resazurin is reduced to C12-resorufin by live cells and is proportional to the number of cells present. Populations can be distinguished by using a flow cytometer with 488 nm and 633 nm laser lines.

Figure 6. Flow cytometric analysis of Jurkat cells using the Metabolic Activity/Annexin V/Dead Cell Apoptosis Kit. Jurkat human T-cell leukemia cells were first exposed to either 10 µM camptothecin or 2 mM hydrogen peroxide for 4 hours at 37°C, 5% CO₂. The cells were then combined, treated with the reagents in the kit and analyzed by flow cytometry. (A) The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations. The cell populations can be evaluated for metabolic activity using (B) the dodecylresorufin fluorescence versus SYTOX Green fluorescence dot plot and (C) the dodecylresorufin fluorescence versus allophycocyanin fluorescence dot plot.

There are some situations where the optimal method for the detection of apoptosis is not annexin V staining. These include assays where cells are sensitive to the high calcium concentrations required for annexin V binding, or assays where phosphatidylserine detection on adherent cells is adversely affected by trypsinization, and assays where washing of samples is prohibitive. We provide unique flow cytometry assays to measure membrane changes under conditions where annexin V binding is problematic.

Learn about additional apoptosis assays for imaging, microplates, or high-content analysis

• No special buffers or wash steps required
• Simple, 5-minute staining protocol for flow cytometry
• Compatible with other blue-excited apoptotic stains
• Accurate apoptotic analysis on trypsinized cells

The Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit provides an easy, efficient method for the detection of apoptosis with dead cell discrimination using a violet laser flow cytometer. Unlike annexin-based assays, this assay does not require special buffers or wash steps, and it is less susceptible to the cell membrane damage commonly found during the physical or chemical removal steps when assaying adherent cells, therefore providing better data quality. The Violet Ratiometric Membrane Asymmetry Probe (F2N123S) works well on adherent and suspension cells and correlates with other indicators of apoptosis, such as caspase detection and changes in mitochondrial membrane potential.

The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. The F2N12S probe is combined with SYTOX AADvanced dead cell stain, which is capable of passing through the cell membrane only in late apoptotic or necrotic cells allowing discrimination form early apoptotic cells (Figure 7). The Violet Ratiometric Membrane Asymmetry Probe⁄Dead Cell Apoptosis Kit can also be paired with MitoProbe DilC1(5) for analysis of apoptosis and viability (Figure 8).

Figure 7. Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection. Jurkat cells (T-cell leukemia, human) were treated with 10 μM camptothecin for four hours (panels B and D) or left untreated as a control (panels A and C). Samples were analyzed on a flow cytometer with 405 nm excitation using 585 nm and 530 nm bandpass filters for F2N12S, and 488 nm excitation for SYTOX AADvanced dead cell stain using a 695 nm bandpass filter. Living cells can be discriminated from apoptotic and dead cells by the relative intensities of the two emission bands from F2N12S (A and B). In panels C and D, SYTOX AADvanced dead cell stain fluorescence is plotted against a derived ratio parameter from the two emission bands (585/530 nm) of F2N12S. A = apoptotic cells, L = live cells, D = dead cells.

Figure 8. Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit for apoptosis detection. Control (top row) and 4-hour camptothecin-treated (bottom row) Jurkat cells were stained with 1 µM SYTOX® AADvanced™ Dead Cell Stain, 200 nM F2N12S, and 50 nM MitoProbe™ DiIC1(5). Dead cells were first excluded (histograms) by gating live cells (those that have lower SYTOX® AADvanced™ fluorescence, indicated by bars). Bivariant density plots show a two-parameter apoptosis assay for mitochondrial membrane potential loss (decreased DiIC1(5) fluorescence) and breakdown of membrane asymmetry (smaller F2N12S 585/530 nm fluorescence ratio). The A, L, and D labels on the graphs indicate apoptotic, live, and dead cells, respectively.