BD Horizon™ BUV395 Annexin V
(RUO)
Preparation And Storage
Recommended Assay Procedures
BD Horizon BUV395 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. BUV395 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the BUV395 Annexin V Staining Protocol. Investigators should note that BUV395 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 BUV395 Annexin V may be used on a cell line (Jurkat).
Materials
1. Prepare Camptothecin stock solution (Sigma-Aldrich Cat. No. C-9911): 1 mM in DMSO.
2. Jurkat T cells (ATCC TIB-152).
Procedure
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 BUV395 Annexin V Staining Protocol to measure apoptosis.
Reagents
1. BD Horizon BUV395 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 BUV395 ANNEXIN V STAINING PROTOCOL
Staining
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 BUV395 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 BUV395 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no BUV395 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 BUV395 Annexin V alone or with BUV395 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 (BUV395 Annexin V positive, 7-AAD negative or BUV395 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 BUV395 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 BUV395 Annexin V and 7-AAD.
Product Notices
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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™ BUV395. 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, BUV395 Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.
BUV395 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 BUV395 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 (BUV395 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 BUV395 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are BUV395 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both BUV395 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 BUV395 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being BUV395 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to BUV395 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to BUV395 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 BUV395 Annexin V and 7-AAD positive, by itself reveals less information about the process by which the cells underwent their demise.
The Annexin V was conjugated to BD Horizon BUV395 which is part of the BD Horizon Brilliant™ Ultraviolet family of dyes. This dye is optimal for multicolor flow cytometry because it has little to no spillover into other detectors. With an Ex Max at 348 nm and an Em Max at 395 nm, BD Horizon BUV395 can be excited with a 355 nm laser and detected with a 379/28 filter.
When compensating dyes in this spectral range, the most accurate compensation can be obtained using unstained and single color-stained cellular controls.
Development References (8)
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Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem. 1990; 265(9):4923-4928. (Biology). View Reference
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Casciola-Rosen L, Rosen A, Petri M, Schlissel M. Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1996; 93(4):1624-1629. (Methodology: Apoptosis, Flow cytometry). View Reference
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Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger CP, Roos D. Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood. 1995; 85(2):532-540. (Biology). View Reference
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Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994; 84(5):1415-1420. (Methodology: Apoptosis, Flow cytometry). View Reference
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Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995; 182(5):1545-1556. (Biology). View Reference
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Raynal P, Pollard HB. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994; 1197(1):63-93. (Biology). View Reference
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Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995; 184(1):39-51. (Methodology: Apoptosis, Flow cytometry). View Reference
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van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry. 1996; 24(2):131-139. (Methodology: Apoptosis, Flow cytometry). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.
