Annexin-V Staining

Alterations in the plasma membrane are a hallmark indicator of early-stage apoptosis in living cells. During the initial phases of apoptosis, phosphatidylserine (PS) residues translocate from the inner to the outer leaflet of the plasma membrane. In cell imaging and flow cytometry, fluorescently labeled annexin V conjugates are commonly used to detect apoptotic cells by their ability to bind to externalized PS residues.

Fig. 1

Annexin V-FITC was used to identify levels of apoptosis in HeLa cells. HeLa cells were treated with low concentrations of EtOH to induce apoptosis and then stained with annexin V-FITC. The green label on the plasma membrane (Annexin V-FITC) indicates cells undergoing apoptosis.

Annexin V

Annexins are a family of calcium-dependent phospholipid-binding proteins. They are found in abundance in eukaryotic organisms (animal, plant, and fungi), where they play critical roles in various cellular and physiological processes such as signal transduction, vesicle transport, and membrane scaffolding. Annexin V specifically is a 35 kDa phospholipid-binding protein. Its strong calcium-dependent affinity for phosphatidylserine (PS) can be used to identify cells undergoing apoptosis. In viable cells, negatively charged PS residues are located on the cytosolic surface of the plasma membrane. However, during early apoptosis, cells lose plasma membrane asymmetry, and PS residues translocate to the outer leaflet of the membrane, where fluorescently labeled annexin V conjugates can detect them.

Annexin V Conjugates for Detecting Apoptosis

Annexin V conjugated to iFluor® dyes provides a fast and reliable method for detecting externalized PS, a key characteristic in early-stage apoptosis. The superior brightness and photostability of Annexin V iFluor® probes significantly outperform most Alexa Fluor® and other spectrally similar annexin v conjugates. Annexin V iFluor® conjugates are useful for live-cell imaging, immunofluorescence, and flow cytometry. They can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necrotic, and dead cells.

Fig. 2

Jurkat cells were treated with 1 µM staurosporine for 4 hours to induce apoptosis. Following treatment, cells were stained with Annexin V-iFluor^® 555 conjugate (red). The nuclei of dead cells were labeled using Nuclear Green™ DCS1 (green). Images were acquired on a confocal microscope.

AAT Bioquest offers a wide variety of fluorescently labeled annexin V conjugates in colors ranging from UV to infrared for improved multiplexing flexibility. We also offer recombinant annexin V and easy-to-use kits optimized for flow cytometry, as well as biotinylated annexin V, which can be detected using fluorescently labeled streptavidin.

ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
N/D = not determined.

Annexin V Conjugates for Flow Cytometry

Annexin V conjugated to mFluor™ dyes, PacBlue, APC and PE are valuable tools for measuring apoptosis by flow cytometry. These conjugates can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necrotic, and dead cells. We offer annexin V conjugates for flow cytometry as stand-alone reagents or easy-to-use kits.

Fig. 3

The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 µM staurosporine (Red) at 37°C for 4 hours, and then ed with Annexin V-mFluor™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.

Key Features of Annexin V mFluor™ Conjugates

Superior brightness and improved photostability for long-term cellular imaging
pH-insensitive fluorescence over a wide molar range
Large Stokes shifts to reduce cross-talk between the excitation source and fluorescence emission for cellular imaging with high signal-to-noise ratios
Compatible with live cells only
Fixable after staining with formaldehyde
Conjugates are efficiently excited by common lasers for increased multiplexing capabilities

ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).

Cell Meter™ Annexin V Binding Apoptosis Assays

Cell Meter™ Annexin V Binding Apoptosis Assay Kits provide a fast and convenient method for measuring apoptotic cells based on the externalization of phosphatidylserine. Each kit contains a recombinant annexin V conjugated to either an iFluor® dye, mFluor™ dye, APC, PE, or FITC and used to detect translocated PS in cells undergoing early apoptosis. In addition, these kits (excluding 22825, 22826, and 22827) include a ready-to-use solution of propidium iodide (PI) or Nuclear Red™ DCS1 nucleic acid stain. Both PI and Nuclear Red™ DCS1 are impermeant to live cells but stain necrotic and dead cells with intense red fluorescence. After staining cells with annexin V conjugate and nucleic acid stain, apoptotic cells show annexin V conjugate fluorescence, dead cells show red and annexin V conjugate fluorescence, and live cells show little or no fluorescence. Cell populations can readily be differentiated using a flow cytometer.

Fig. 4

Key Features of Cell Meter™ Annexin V Binding Apoptosis Assays

High Sensitivity - annexin V labeled with iFluor®, or mFluor™ dyes produce significantly brighter and more photostable signals than Alexa Fluor and other spectrally similar annexin V conjugates
Flexibility - wide range of fluorescently labeled annexin V conjugates to match any instrument set-up
Convenient design - streamlined assay procedure which can be completed in 30 to 60 minutes

For Cat No. 22825, 22826 and 22827 use Nuclear Blue™ DCS1 as a dead cell stain.

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Document: 01.0077.211015r1

Last updated Thu Aug 28 2025