Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry
Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry
Invitrogen™

Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violet-fluorescent Pacific Blue™ dyeRead more
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Catalog number A35136
Price (USD)
542.00
Each
Add to cart
Price (USD)
542.00
Each
Add to cart
This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violet-fluorescent Pacific Blue™ dye and dead cells using SYTOX™ AADvanced™ stain. After staining a cell population with Pacific Blue™, annexin V and SYTOX™ AADvanced™, apoptotic cells show violet fluorescence, dead cells show red fluorescence, and live cells show little or no fluorescence. These populations are easily distinguished by a flow cytometer with the 405 nm and 488 nm lines for excitation. There is very little spectral overlap between the two dyes, therefore very little compensation is needed. Each kit contains sufficient reagents for approximately 50 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Quantity1 kit
FormatTube
Product TypeApoptosis Kit
Flow Cytometer Laser Lines405, 488
Excitation/EmissionPacific Blue™: 415⁄455, SYTOX™ AADvanced™: 546⁄647
No. of Reactions50
Shipping ConditionWet Ice
For Use With (Application)Flow Cytometry
ConjugatePacific Blue, SYTOX AADvanced Dead Cell Stain
For Use With (Equipment)Flow Cytometer
Unit SizeEach
Contents & Storage
Contains 1 vial of annexin V, Pacific Blue™ conjugate (250 μL), 1 vial of SYTOX™ AADvanced™ dead cell stain, and 1 bottle of annexin binding buffer (5X solution, 15 mL), and 1 vial of DMSO (100 μL).

Store in refrigerator (2–8°C) and protect from light.

Frequently asked questions (FAQs)

I want to study apoptosis using an Annexin V conjugate, but with adherent cells via microscopy instead of flow cytometry. Can this be done?

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

I trypsinized my adherent cells and labeled with annexin V, and now my flow data is showing a high percentage of apoptotic cells even for control, untreated cells. What is the problem?

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).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can I detect annexin V staining in an imaging assay?

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

When should I stain adherent cells with annexin V for flow cytometric analysis? Before or after I trypsinize them?

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).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can I fix my cells after annexin V labeling?

Annexin V staining is best analyzed on live cells. If you need to fix your cells for analysis, then fix in 3.7% formaldehyde in PBS containing calcium and magnesium to maintain binding during fixation. The signal will not be retained after permeabilization, thus annexin V staining is not compatible with internal antibody labeling.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.