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.

Annexin V

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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 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

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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.

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.

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

Annexin V Conjugates for Flow Cytometry

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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.

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

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

Cell Meter™ Annexin V Binding Apoptosis Assays

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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.

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

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

Product Ordering Information

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