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.
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.
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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).
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
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.
Flow cytometry experiment using the Metabolic Activity 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.
Citations
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 | A23204 |
| 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.
| 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.
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 | A23204 |
| 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. 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.
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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