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Molecular Probes™ Annexin V Conjugates for Apoptosis Detection
Description
Achieve quick and reliable detection of early cell apoptosis with Annexin V stand-alone conjugates for apoptosis detection. Annexin V conjugates offer up to 100-fold difference in fluorescence signal intensity between apoptotic and non-apoptotic cells using flow cytometry.
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.
In collaboration with Nexins Research BV, we provide the best and brightest annexin V conjugates available, including Alexa Fluor 350, 488, 555, 568, 594, 647, and 680 annexin V conjugates, as well as Annexin V APC, Biotin-X, FITC, Pacific Blue, and PE conjugates. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, one of the earliest indicators of apoptosis.
The Annexin V Pacific Blue conjugate is violet excitable, making it ideal for instruments with a violet laser and for multicolor experiments that include green- or red-fluorescent dyes.
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
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.
Specifications
Specifications
| Description | Annexin V, Alexa Fluor 488 conjugate (replacement for Cat. No. PHN1010 and PHN1008) |
| Quantity | 500 μL |
| Kit Contents | Contains 1 vial of annexin V, Alexa Fluor 488 conjugate. |
| Product Type | Annexin V conjugate |
| Content And Storage | Store in refrigerator (2°C to 8°C) and protect from light. |
| Flow Cytometer Laser Lines | 488 |
| Excitation/Emission | 495/519 |
| No. of Reactions | 100 |
| Color | Green |
| Shipping Condition | Wet Ice |
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Frequently Asked Questions (FAQs)
It has been done, but we don‘t recommend it. Both healthy cells and apoptotic cells possess phosphatidylserine on the cell surface, which can be detected with Annexin V, but apoptotic cells have significantly more of it. You can easily tell the difference between these two populations with flow cytometry, because flow cytometers are more sensitive and have a higher throughput. But with a microscope, you cannot always tell the difference, especially for adherent cells. Instead, for microscopy, we recommend a different technique, such as detecting caspases with CellEvent Caspase Detection Reagents.
Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. Allow the cells to recover for about 30 minutes in optimal cell culture conditions and medium after trypsinizing/scraping so that they can recover their membrane integrity before staining. For lightly adherent cell lines, such as HeLa and NIH 3T3, another option is to use non-enzyme treatments like Gibco Cell Dissociation Buffer (Cat. No. 13151014).
Annexin V staining is not typically used in imaging experiments; it is a better reagent for flow cytometry analysis. All cells will stain to some extent, so it can be difficult to distinguish a relatively bright annexin V-stained cell from a dimmer non-apoptotic cell. Caspase activation, detected using our CellEvent Caspase 3/7 or Image-iT LIVE Caspase detection kits, is a better method for detecting apoptosis in an imaging assay.
Trypsinize first and then allow the cells to recover about 30 minutes in optimal cell culture conditions and medium before staining with annexin V conjugates. Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing for annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. For lightly adherent cell lines such as HeLa and NIH 3T3, you could use a less harsh (non-enzymatic) dissociation product like Gibco Cell Dissociation Buffer (Cat. No. 13151014).
Yes, this is possible. We have established protocols for annexin V staining combined with intracellular staining of lymphocytes that can be found here. The most important step is to leave some binding buffer in the suspension when fixation is started. Compared to staining of live cells, the intensity of the annexin V signal may be somewhat reduced.
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