BV711 Annexin V
- Brand BD Horizon™
- Reactivity Human (QC Testing)
Flow cytometry (Routinely Tested)
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
Apoptosis is a normal physiologic process that occurs during embryonic development as well as in maintenance of tissue homeostasis. The apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma membrane asymmetry is one of the earliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including BD Horizon™ BV711. This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, BV711Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.
BV711 Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with BV711 Annexin V is typically used in conjunction with a vital dye such as 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (BV711 Annexin V positive, 7-AAD negative). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to the nucleic acid dye, 7-AAD. For example, cells that are considered viable are both BV711 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are BV711 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both BV711 Annexin V positive and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both BV711 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being BV711 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to BV711 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to BV711 Annexin V positive and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both BV711 Annexin V and 7-AAD positive, by itself reveals less information about the process by which the cells underwent their demise.
The Annexin V is conjugated to BD Horizon BV711 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 711-nm. BD Horizon BV711 can be excited by the violet laser and detected in a filter used to detect Cy™5.5 / Alexa Fluor® 700-like dyes (eg, 712/20-nm filter). Due to the excitation and emission characteristics of the acceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy5.5 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
When compensating dyes in this spectral range, the most accurate compensation can be obtained using unstained and single color-stained cellular controls.
BV711 is a tandem fluorochrome of BD Horizon BV421 and an acceptor dye with an emission maximum at 711 nm. This dye offers a very bright choice for the violet laser. Due to the excitation and emission characteristics of the acceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy5.5 detectors. BV711 will also have moderate spillover into the BD Horizon Brilliant™ Violet 786 detector.
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- BD Horizon Brilliant Violet 711 is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,227,187; 8,455,613; 8,575,303; 8,354,239.
- BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
BD Horizon BV711 Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces with a higher affinity for phosphatidylserine (PS) than most other phospholipids. BV711 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the BV711 Annexin V Staining Protocol. Investigators should note that BV711 Annexin V flow cytometric analysis on adherent cell types (eg, HeLa, NIH-3T3, etc.) is not routinely tested as specific membrane damage may occur during cell detachment or harvesting. Methods for utilizing Annexin V for flow cytometry on adherent cell types, however, have been previously reported (Casiola-Rosen et al. and van Engelend et al.).
INDUCTION OF APOPTOSIS BY CAMPTOTHECIN
The following protocol is provided as an illustration on how BV711 Annexin V may be used on a cell line (Jurkat).
1. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. No. C-9911): 1 mM in DMSO.
2. Jurkat T cells (ATCC TIB-152).
1. Add Camptothecin (final conc. 4-6 µM) to 1 × 10^6 Jurkat cells.
2. Incubate the cells for 4-6 hr at 37°C.
3. Proceed with the BV711 Annexin V Staining Protocol to measure apoptosis.
1. BD Horizon BV711 Annexin V: Included. Use 5 µl per test.
2. 7-Amino-Actinomycin D (7-AAD): Not included. 7-AAD (Cat. No. 559925) is a convenient, ready-to-use nucleic acid dye with fluorescence detectable in the far red range of the spectrum. Use 5 µl per test.
3.10X Annexin Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, BD Pharmingen™ Annexin V Binding Buffer, 10X concentrate (Cat. No. 556454) may be purchased.
BD Horizon BV711 ANNEXIN V STAINING PROTOCOL
1. Wash cells twice with cold PBS and then resuspend cells in 1× Binding Buffer at a concentration of 1 × 10^6 cells/ml.
2. Transfer 100 µl of the cell suspension (1 × 10^5 cells) to a 5 ml culture tube.
3. Add 5 µl of BV711 Annexin V (for one and two color analysis) and 5 µl of 7-AAD (for two color analysis only).
4. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.
5. Add 400 µl of 1× Annexin V Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY
The following controls are used to set up compensation and quadrants:
1. Unstained cells.
2. Cells stained with BV711 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no BV711 Annexin V).
Other Staining Controls:
A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with BV711 Annexin V alone or with BV711 Annexin V and 7-AAD. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis (BV711 Annexin V positive, 7-AAD negative or BV711 Annexin V positive, 7-AAD positive).
The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated population from the percentage of apoptotic cells in the treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged membrane and stain positive for 7-AAD as well as for BV711 Annexin V. Thus, the assay does not distinguish between cells that have already undergone an apoptotic cell death and those that have died as a result of necrotic pathway, because in either case the dead cells will stain with both BV711 Annexin V and 7-AAD.