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
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.
Table 1. Overview of annexin V conjugates.
Specification ▲ ▼ | Value ▲ ▼ |
Application | Detects externalized phosphatidylserine to identify apoptotic cells |
Readout | Fluorescence microscopy, live cell imaging and flow cytometery |
Live Cells | Yes |
Fixed Cells | Not for use |
Fixable | Yes, fix after staining with formaldehyde |
Multiplexing | Yes, bright signals and broad wavelength ranges for multiplex analysis |
Annexin V Conjugates for Detecting Apoptosis

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.
Table 2. Available annexin V conjugates for apoptosis detection.
Annexin V Label ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Filter Set ▲ ▼ | ε¹ ▲ ▼ | Φ² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
iFluor® 350 | 345 | 450 | DAPI | 20000 | 0.95 | 100 tests (200 µL) | 20070 |
iFluor® 488 | 491 | 516 | FITC | 75000 | 0.9 | 100 tests (200 µL) | 20071 |
iFluor® 555 | 557 | 570 | TRITC | 100000 | 0.64 | 100 tests (200 µL) | 20072 |
iFluor® 594 | 588 | 604 | Texas Red | 180000 | 0.53 | 100 tests (200 µL) | 20073 |
iFluor® 633 | 640 | 654 | Texas Red | 250000 | 0.29 | 100 tests (200 µL) | 20069 |
iFluor® 647 | 656 | 670 | Cy5 | 250000 | 0.25 | 100 tests (200 µL) | 20074 |
iFluor® 680 | 684 | 701 | Cy5.5 | 220000 | 0.23 | 100 tests (200 µL) | 20075 |
iFluor® 700 | 690 | 713 | Cy5.5 | 220000 | 0.23 | 100 tests (200 µL) | 20077 |
iFluor® 750 | 757 | 779 | Cy7 | 275000 | 0.12 | 100 tests (200 µL) | 20076 |
XFD350 (Alexa Fluor® 350 equivalent) | 343 | 441 | DAPI | 19000 | N/D³ | 100 tests (200 µL) | 20090 |
- 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

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
Table 3. Available annexin V conjugates for optimized for flow cytometry.
Annexin V Label ▲ ▼ | Laser ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Channel ▲ ▼ | ε¹ ▲ ▼ | Φ² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
PacBlue | Violet Laser | 410 | 455 | DAPI/FL-1 Channel | 46000 | 0.78 | 100 tests (200 µL) | 20089 |
mFluor™ Violet 450 | Violet Laser | 403 | 454 | DAPI/FL-1 Channel | 25000 | 0.92 | 100 tests (200 µL) | 20080 |
mFluor™ Violet 510 | Violet Laser | 414 | 510 | FITC/FL-1 Channel | 30000 | 0.86 | 100 tests (200 µL) | 20081 |
mFluor™ Violet 540 | Violet Laser | 424 | 560 | PE/FL-2 Channel | 15000 | 0.64 | 100 tests (200 µL) | 20082 |
mFluor™ Blue 570 | Blue Laser | 533 | 570 | PE/FL-2 Channel | 120000 | 0.08 | 100 tests (200 µL) | 20085 |
- 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.

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 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
Table 4. Cell Meter™ Annexin V Binding Apoptosis Assay Selection Guide
Annexin V Conjugate ▲ ▼ | Dead Cell Stain ▲ ▼ | Annexin V Conjugate Ex/Em ▲ ▼ | Dead Cell stain Ex/Em ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
Annexin V-iFluor® 488 | PI | 490/ 520 nm | 534/617 nm | 100 Tests | 22824 |
Annexin V-iFluor® 555 | None | 554/578 nm | *** | 100 Tests | 22825 |
Annexin V-iFluor® 594 | None | 590/610 nm | *** | 100 Tests | 22826 |
Annexin V-iFluor® 647 | None | 650/668 nm | *** | 100 Tests | 22827 |
Annexin V-mFluor™ Violet 450 | PI | 405/450 nm | 534/617 nm | 100 Tests | 22828 |
Annexin V-mFluor™ Violet 500 | PI | 414/508 nm | 534/617 nm | 100 Tests | 22829 |
Annexin V-mFluor™ Violet 550 | PI | 424/560 nm | 534/617 nm | 100 Tests | 22830 |
Annexin V-APC | PI | 651/662 nm | 534/617 nm | 100 Tests | 22837 |
Annexin V-PE | Nuclear Red™ DCS1 | 565/575 nm | 631/651 nm | 100 Tests | 22838 |
Annexin V-FITC | PI | 490/520 nm | 534/617 nm | 100 Tests | 22839 |
- For Cat No. 22825, 22826 and 22827 use Nuclear Blue™ DCS1 as a dead cell stain.
Product Ordering Information
Table 5. Ordering information for recombinant annexin V, annexin V conjugates, and annexin V assay kits.
Cat No. ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
20014 | Annexin V, Recombinant | 100 ug |
20015 | Annexin V, Recombinant | 1 mg |
20018 | Annexin V-Biotin conjugate | 100 tests |
20030 | Annexin V, FITC Labeled | 100 tests |
20031 | Annexin V, TRITC Labeled | 100 tests |
20065 | Annexin V-Cy3 conjugate | 100 tests |
20066 | Annexin V-Cy5 conjugate | 100 tests |
20067 | Annexin V-Cy5.5 conjugate | 100 tests |
20068 | Annexin V-Cy7 conjugate | 100 tests |
20069 | Annexin V-iFluor® 633 conjugate | 100 tests |