Microscopic view of apoptotic cells with green and orange fluorescence

Annexin V staining is a common method for detecting apoptotic cells. Thermo Fisher Scientific offers high-quality fluorescent annexin V conjugates as standalone reagents and in a variety of kits for use in flow cytometry and for imaging suspension cells.

See selection guide

Why is annexin V staining used to detect apoptosis?

Fluorescent conjugates of annexin V are commonly used to identify apoptotic cells. The human vascular anticoagulant annexin V is a 35–36 kDa, Ca2+-dependent phospholipid-binding protein that has a high affinity for the anionic phospholipid phosphatidylserine (PS). In normal healthy cells, PS is located on the cytoplasmic surface of the plasma membrane. However, during apoptosis, the plasma membrane undergoes structural changes that include translocation of PS from the inner to the outer leaflet (extracellular side) of the plasma membrane. It has been reported that the translocated phosphatidylserine on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages (1).

Illustration showing cellular location of PS location normal and apoptotic cells, with annexin v binding externalized PS

Annexin V conjugates for apoptosis detection

We have collaborated with Nexins Research BV—the original developer of fluorescent phosphatidylserine-binding proteins—to produce annexin V conjugates with superior brightness. These annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained with our fluorescent annexin V conjugates, as measured by flow cytometry, is typically about 100-fold.

The benefits of our annexin V conjugates include:

  • Conjugated to Invitrogen Alexa Fluor and eFluor dyes for brighter signals
  • Conjugates for all available lasers
  • Available as stand-alone reagents or easy-to-use kits

Experimental conditions for annexin V staining

Annexin V staining to detect apoptotic cells can only be done on live cells and tissue. If samples are to be fixed post-staining, there are specific conditions required to achieve transient retention of signal. These include use of an alcohol-free, aldehyde-based fixation method, use of buffers containing Ca2+ and avoidance of surfactants/detergents. For your convenience, we also offer a concentrated annexin-binding buffer that facilitates the binding of annexin V to phosphatidylserine in apoptosis assays.

Avoiding false positives

It should be noted that there is a risk of false positives when staining with annexin V conjugates. Compromised plasma membranes of dead cells provide a path for annexin V protein to pass through to the interior of the cell where it can bind PS in the inner leaflet. We recommend using a live cell-impermeant stain in combination with annexin V conjugates. This combination allows for the separation of apoptotic cells from dead cells. While cells positive for both probes may indicate late stage apoptosis, annexin V staining in this population may be due to inner leaflet PS binding and therefore may not be reliably deemed apoptotic. In this combination, cells that are only annexin V-positive and therefore have intact plasma membranes, are demonstrably apoptotic. We offer a range of kits that include an annexin V conjugate and a compatible live cell-impermeant stain (see selection guide). These kits have been optimized for flow cytometry.

Annexin V staining in action

Alexa Fluor 488 Annexin V staining in flow cytometry experiment

Flow cytometry experiment using the Metabolic Activity Dead Cell Apoptosis Kit

3 dot plots of live, dead and apoptotic cells stained with the Metabolic Activity/Annexin V/Dead Cell Apoptosis Kit.

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.

Selection guide for annexin V products

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

    Microscopy Flow cytometry  
Annexin V conjugate Ex/Em (nm) Common emission filters Laser Common emission filters Cat. No.
Alexa Fluor 350 346/442 DAPI UV 450/40 nm A23202
Pacific Blue 410/455 NA 405/7 nm 450/50 nm A35122
Alexa Fluor 488 490/525 FITC 488 nm 530/30 nm A13201
FITC 490/525 FITC 488 nm 530/30 nm A13199
PE 565/578 TRITC 488 nm
532 nm
561 nm
585/42 nm A35111
Alexa Fluor 555 555/580 TRITC 532 nm
561 nm
575/26 nm A35108
Alexa Fluor 568 578/603 Texas Red 532 nm
561 nm
610/20 nm A13202
Alexa Fluor 594 590/617 Texas Red 532 nm 630/20 nm A13203
Alexa Fluor 647 650/665 Cy5 633-637 nm 661/8 nm A13204
APC 650/660 Cy5 633-637 nm 661/8 nm A35110
Alexa Fluor 680 679/702 Cy5.5 633-637 nm 720/30 nm A35109
Biotin-X NA NA NA NA A13204
Required buffer
Annexin Binding Buffer (5x) V13246

Alternatives to annexin V staining

There are some situations where staining cells with annexin V is not the optimal method for the detection of apoptosis. 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.

When the use of calcium containing buffers isn’t an option

  • 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 is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. It 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.

Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection

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.

Product Laser Ex/Em Apoptotic cell stain Dead cell stain Cat. No.
Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit 405 and 488 nm 405/585 (live),
405/530 (apoptotic),
546/647 (dead)
F2N12S SYTOX AADvanced A35137
Membrane Permeability/Dead Cell Apoptosis Kit 405 and 488 nm 434/456 nm (apoptotic)
546/647 nm (dead)
PO-PRO-1 7-AAD V35123

Alternatives ways to assess cell apoptosis

Although annexin V conjugates are widely used, there are additional methods for assessing cells for apoptosis.

Learn more about other Apoptosis Assays

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

Annexin V conjugate Dead cell stain Approximate fluorescence excitation/emission maxima Additional reagents in kit Size Cat. No.
Annexin V conjugate Dead cell stain
Annexin V, eFluor 450 7-AAD 405/450 nm 546/647 nm
Annexin binding buffer (10x) 50 assays 88-8006-72
200 assays 88-8006-74
Annexin V, Pacific Blue SYTOX AADvanced 415/455 nm 546/647 nm Annexin binding buffer (5x) 50 assays A35136
Annexin V, Alexa Fluor 488 PI 499/521 nm 535/617 nm Annexin binding buffer (5x) 50 assays V13241
250 assays V13245
Annexin V, Alexa Fluor 488 SYTOX Green 499/521 nm 503/524 nm Annexin binding buffer (5x) 50 assays V13240
Annexin V, Alexa Fluor 488 none 499/521 nm NA • MitoTracker Red (CMXRos)
• Annexin binding buffer (5x)
• DMSO
50 assays V35116
Annexin V, Fluorescein PI 494/518 nm 535/617 nm Annexin binding buffer (5x) 50 assays V13242
Annexin V, PerCP-eFluor 710 482/710 nm NA Annexin binding buffer (10x) 50 assays 88-8008-72
200 assays 88-8008-74
Annexin V, APC SYTOX Green 650/660 nm 503/524 nm Annexin binding buffer (5x) 50 assays V35113
Annexin V, RPE SYTOX Green 488/575 nm 503/524 nm Annexin binding buffer (5x) 50 assays V35112
Annexin V, RPE-Cyanine7 488/767 nm NA Annexin binding buffer (10x) 50 assays 88-8103-72
200 assays 88-8013-74
Annexin V, APC SYTOX Green 650/660 nm 503/524 nm • Annexin binding buffer (5x)
• C12-resazurin (571/585 nm)
50 assays V35114

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

    Microscopy Flow cytometry  
Annexin V conjugate Ex/Em (nm) Common emission filters Laser Common emission filters Cat. No.
Alexa Fluor 350 346/442 DAPI UV 450/40 nm A23202
Pacific Blue 410/455 NA 405/7 nm 450/50 nm A35122
Alexa Fluor 488 490/525 FITC 488 nm 530/30 nm A13201
FITC 490/525 FITC 488 nm 530/30 nm A13199
PE 565/578 TRITC 488 nm
532 nm
561 nm
585/42 nm A35111
Alexa Fluor 555 555/580 TRITC 532 nm
561 nm
575/26 nm A35108
Alexa Fluor 568 578/603 Texas Red 532 nm
561 nm
610/20 nm A13202
Alexa Fluor 594 590/617 Texas Red 532 nm 630/20 nm A13203
Alexa Fluor 647 650/665 Cy5 633-637 nm 661/8 nm A13204
APC 650/660 Cy5 633-637 nm 661/8 nm A35110
Alexa Fluor 680 679/702 Cy5.5 633-637 nm 720/30 nm A35109
Biotin-X NA NA NA NA A13204
Required buffer
Annexin Binding Buffer (5x) V13246

Alternatives to annexin V staining

There are some situations where staining cells with annexin V is not the optimal method for the detection of apoptosis. 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.

When the use of calcium containing buffers isn’t an option

  • 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 is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. It 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.

Violet Ratiometric Membrane Asymmetry Probe for apoptosis detection

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.

Product Laser Ex/Em Apoptotic cell stain Dead cell stain Cat. No.
Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit 405 and 488 nm 405/585 (live),
405/530 (apoptotic),
546/647 (dead)
F2N12S SYTOX AADvanced A35137
Membrane Permeability/Dead Cell Apoptosis Kit 405 and 488 nm 434/456 nm (apoptotic)
546/647 nm (dead)
PO-PRO-1 7-AAD V35123

Alternatives ways to assess cell apoptosis

Although annexin V conjugates are widely used, there are additional methods for assessing cells for apoptosis.

Learn more about other Apoptosis Assays

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

Annexin V conjugate Dead cell stain Approximate fluorescence excitation/emission maxima Additional reagents in kit Size Cat. No.
Annexin V conjugate Dead cell stain
Annexin V, eFluor 450 7-AAD 405/450 nm 546/647 nm
Annexin binding buffer (10x) 50 assays 88-8006-72
200 assays 88-8006-74
Annexin V, Pacific Blue SYTOX AADvanced 415/455 nm 546/647 nm Annexin binding buffer (5x) 50 assays A35136
Annexin V, Alexa Fluor 488 PI 499/521 nm 535/617 nm Annexin binding buffer (5x) 50 assays V13241
250 assays V13245
Annexin V, Alexa Fluor 488 SYTOX Green 499/521 nm 503/524 nm Annexin binding buffer (5x) 50 assays V13240
Annexin V, Alexa Fluor 488 none 499/521 nm NA • MitoTracker Red (CMXRos)
• Annexin binding buffer (5x)
• DMSO
50 assays V35116
Annexin V, Fluorescein PI 494/518 nm 535/617 nm Annexin binding buffer (5x) 50 assays V13242
Annexin V, PerCP-eFluor 710 482/710 nm NA Annexin binding buffer (10x) 50 assays 88-8008-72
200 assays 88-8008-74
Annexin V, APC SYTOX Green 650/660 nm 503/524 nm Annexin binding buffer (5x) 50 assays V35113
Annexin V, RPE SYTOX Green 488/575 nm 503/524 nm Annexin binding buffer (5x) 50 assays V35112
Annexin V, RPE-Cyanine7 488/767 nm NA Annexin binding buffer (10x) 50 assays 88-8103-72
200 assays 88-8013-74
Annexin V, APC SYTOX Green 650/660 nm 503/524 nm • Annexin binding buffer (5x)
• C12-resazurin (571/585 nm)
50 assays V35114

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