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 imaging and flow cytometry instrument platforms.

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 cell membrane. However, during the apoptosis process, the cell membrane undergoes structural changes that include translocation of PS from the intracellular side of the plasma membrane to the outer leaflet (extracellular side) of the plasma membrane. It is thought 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.

It should also be noted that while annexin V conjugates bind to PS on apoptotic cell surfaces, the protein can also pass through the compromised membranes of dead cells and bind to PS in the interior of the cell (2). Therefore, we recommend using a cell-impermeant dead cell stain in combination with annexin V conjugate staining to distinguish dead cells from apoptotic cells. We offer a range of kits that include an annexin V conjugate and a compatible cell-impermeant nucleic acid stain (see selection guide below). These kits have been optimized for flow cytometry but can be used for imaging as well.

See our selection guide for annexin V products

Annexin V staining in action

Alexa Fluor 488 Annexin V staining in flow cytometry experiment

Jurkat cells (T cell leukemia, human) treated with 10 μM camptothecin for 4 hours (right panel) or untreated (as control, left panel). Cells were then treated with Annexin V, Alexa Fluor 488 conjugate to identify apoptotic cells and with propidium iodide to identify dead cells, followed by flow cytometric analysis. Note that the camptothecin-treated cells (right panel) have a higher percentage of apoptotic cells (indicated by an “A”) than the basal level of apoptosis seen in the control cells (left panel). V = viable cells, D = dead cells.

Alexa Fluor 488 Annexin V staining in imaging microscopy experiment

Fluorescence microscope image of green fluorescent apoptotic cells and red fluorescent dead cells

Jurkat human T cell leukemia cells treated with 1 µM camptothecin. The externalized phosphatidylserine, a characteristic of early-stage apoptotic cells, was detected with Annexin V, Alexa Fluor 488 conjugate. The late-stage apoptotic and necrotic cells were stained with propidium iodide. The image was acquired using bandpass filters appropriate for fluorescein.

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

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, 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

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, 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

Resources

BestProtocols: Annexin V staining for Flow Cytometry

Fluorescence SpectraViewer
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