Changes to the plasma membrane are key events during apoptosis. During early apoptosis, phosphatidylserine becomes externalized, which can be recognized by Annexin V staining for flow cytometry.
Design your Annexin V assay
Step 1: Choose Your Annexin V Conjugate
To bring flexibility to your apoptosis workflows, we offer multiple conjugation options to ensure compatibility with different instrument configurations and multicolor panels.
Laser | Annexin V Standalone Conjugate | Cat. No. |
UV | BD Horizon™ BUV395 Annexin V | |
Violet | BD Horizon™ BV421 Annexin V BD Horizon™ V450 Annexin V BD Horizon™ V500 Annexin V BD Horizon™ BV605 Annexin V BD Horizon™ BV711 Annexin V | |
Blue | BD Pharmingen™ PerCP-Cy™5.5 Annexin V BD Pharmingen™ FITC Annexin V | |
Yellow-green | BD Horizon™ PE-CF594 Annexin V * | |
Red | BD Pharmingen™ APC Annexin V BD Pharmingen™ Alexa Fluor™ 647 Annexin V BD Pharmingen™ Cy™5 Annexin V |
*These dyes are also excited by the blue Laser.
Step 2: Choose Your Viability Dye
Annexin V is frequently used with viability dyes. We offer a wide selection of viability dyes that may be used in combination with Annexin V, including PI, 7-AAD, DAPI, Via-Probe™ Green, Via-Probe™ Red and DRAQ7.
See all viability stains
| Laser | Laser Excitation | Product Name | Excitation Max (nm) | Emission Max (nm) | Fixable | Type of Dye | Cat. No. |
|---|---|---|---|---|---|---|---|
| UV 355 nm 40 mW | 355 UV | BD Horizon Fixable Viability Stain 440UV | 355 | 440 | X | Fixable amine-reactive viability dye | 566332 |
| 355 UV / 405 Violet | DAPI Solution | 358 | 461 | DNA intercalating viability dye | 564907 | ||
| Violet 405 nm 100 mW | 405 Violet | BD Horizon Fixable Viability Stain 450 | 406 | 450 | X | Fixable amine-reactive viability dye | 562247 |
| 405 Violet | BD Horizon Fixable Viability Stain 510 | 405 | 510 | X | Fixable amine-reactive viability dye | 564406 | |
| 405 Violet | BD Horizon Fixable Viability Stain 575V | 405 | 575 | X | Fixable amine-reactive viability dye | 565694 | |
| Blue 488 nm 100 mW | 488 Blue | BD Horizon Fixable Viability Stain 520 | 498 | 520 | X | Fixable amine-reactive viability dye | 564407 |
| 488 Blue | Via-Probe Green | 488 | 530 | DNA-binding viability dye | 565799 | ||
| Yellow-Green 561 nm 150 mW | 561 Yellow-Green/488 Blue | Propidium Iodide (PI) Solution | 535 | 617 | DNA-binding viability dye | 556463 | |
| 561 Yellow-Green/488 Blue | BD Via-Probe™ Cell Viability Solution (7-AAD) | 546 | 647 | DNA-binding viability dye | 555816, 559925 | ||
| 561 Yellow-Green | BD Horizon Fixable Viability Stain 570 | 561 | 570 | X | Fixable amine-reactive viability dye | 564995 | |
| 561 Yellow-Green | BD Horizon Fixable Viability Stain 620 | 561 | 620 | X | Fixable amine-reactive viability dye | 564996 | |
| Red 628 nm 200 mW | 640 Red | Via-Probe Red | 633 | 660 | DNA-binding viability dye | 565803 | |
| 640 Red | DRAQ7 | 647 | 697 | DNA-binding viability dye | 564904 | ||
| 640 Red | BD Horizon Fixable Viability Stain 660 | 649 | 660 | X | Fixable amine-reactive viability dye | 564405 | |
| 640 Red | BD Horizon Fixable Viability Stain 700 | 649 | 700 | X | Fixable amine-reactive viability dye | 564997 | |
| 640 Red | BD Horizon Fixable Viability Stain 780 | 649 | 780 | X | Fixable amine-reactive viability dye | 565388 |
Step 3: Choose Your Controls
Every experiment should include both positive (apoptotic cells) and negative (healthy cells) controls to verify assay performance and support accurate interpretation of results. For compensation or spectral unmixing, prepare single-stained controls for the Annexin V conjugate and each additional fluorochrome used in the multicolor panel. These controls should be stained and acquired under conditions that closely match those of the experimental samples to ensure optimal compensation or unmixing accuracy.
Annexin V Detection Kits
In addition to standalone reagents for multicolor panels, we offer Annexin V stains as part of ready-to-use kits. Detection kits consist of pre-paired Annexin V dyes with viability dyes and buffers.
Product | Conjugate | Viability Dye | Cat. No. |
FITC Annexin V Apoptosis Detection Kit I | FITC | PI | |
FITC Annexin V Apoptosis Detection Kit II | FITC | 7-AAD | |
PE Annexin V Apoptosis Detection Kit I | PE | 7-AAD |
Is Annexin V the Right Marker for Your Assay?
Annexin V specifically detects phosphatidylserine externalization, making it a valuable tool for identifying cells in early apoptosis. However, it may not be the optimal choice for every application. If your experimental goals require measuring a different stage of cell death, understanding the mechanism of apoptosis, assessing mitochondrial health, or preserving cell function in calcium-sensitive systems, alternative approaches may be more appropriate. Consider the following options:
- Caspase activation assays (active caspase-3/7 antibodies and Live Cell Caspase Probes) for confirming apoptotic pathway mechanism
- TUNEL assays for DNA fragmentation in late-stage apoptosis
- Standalone nucleic acid dyes (7-AAD, PI, DAPI, Via-ProbeTM Green, Via-ProbeTM Red and DRAQ7) when only live/dead discrimination is needed
- Mitochondrial depolarization dyes (Mitostatus TMRE and Red) detect changes in mitochondrial membrane potential, an early marker of cell stress and apoptosis
- BD HorizonTM Fixable Viability Stains, amine-reactive fluorescent dyes that irreversibly label cells with compromised membrane integrity and are preserved through intracellular workflows
Annexin V staining is straightforward to perform because the assay requires no wash steps.
FAQs
- Which Annexin V conjugate should I use?
Choosing the best Annexin V conjugate starts with your instrument configuration and considering the other fluorochromes or dyes to be included in your panel. As with any fluorochrome assignment, review the spectral profile, brightness, and potential spillover to ensure optimal panel performance. The BD® Spectrum Viewer is a useful tool for evaluating fluorochrome compatibility and selecting the most appropriate Annexin V conjugate for your assay.
- What's the difference between the Annexin V detection kits and standalone conjugates?
Annexin V detection kits provide a convenient, ready-to-use solution for apoptosis detection. Each kit includes: an Annexin V conjugation option (FITC or PE), a viability stain (7-AAD or PI) and an optimized buffer for proper staining of Annexin V (10x Annexin V Binding Buffer). The kits are the perfect option for those searching for a simplified way to detect apoptotic cells and differentiate them from live and dead cells. Standalone Annexin V conjugates are ideal for those looking for more flexible options to incorporate the detection of apoptotic cells in multicolor panels.
- How do I compensate or unmix Annexin V in a multicolor panel?
In a spectral or conventional flow cytometry panel, Annexin V is unmixed or compensated the same way as any other fluorochrome—by providing the instrument with an accurate single-stained reference control for the specific Annexin V conjugate used. Use a highly positive population for the positive reference control. To establish a negative population, use an unstained control of the same cell type.
- Why do I need a viability dye in addition to Annexin V?
Annexin V and viability dyes assess different aspects of cell health. Annexin V serves as a marker of early apoptosis by detecting phosphatidylserine exposed on the outer cell membrane, while viability dyes label cells with compromised membrane integrity, allowing identification of late apoptotic and dead cells. Unlike Annexin V, fixable viability stains require a wash step after staining. As a result, cells that die after the dye has been washed away will not be stained, which may underestimate cell death occurring later in the assay workflow.
- Are Annexin V assays compatible with image-enabled flow cytometry?
Yes. We offer a wide range of options for Annexin V conjugation and viability dyes compatible with conventional, spectral, and image-enabled flow cytometry. See an example of how we used Annexin V to differentiate between live non-apoptotic cells and live apoptotic cells from dead cells using spectral and image-enabled flow cytometry in the Label-free identification of apoptotic cell death poster.
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale.