BD Horizon™ V450 Annexin V

V450

ANNEXIN V

Apoptosis is a normal physiological process that occurs during embryonic development and tissue homeostasis maintenance. The apoptotic program is characterized by certain morphological features, including plasma membrane symmetry and detachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of the plasma membrane is one of the earliest features of apoptosis. In apoptotic cells, phosphatidylserine (PS), a phospholipid membrane component, 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™ V450. 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, V450 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation.   V450 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 V450 Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, V450 Annexin V positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. For example, cells that are considered viable are both V450 Annexin V and PI negative, cells that are in early apoptosis are V450 Annexin V positive and PI negative, and cells that are in late apoptosis or already dead are both V450 Annexin V and PI positive. This assay does not distinguish between cells that have undergone the apoptotic versus necrotic pathway because dead cells will be stained with both V450 Annexin V and PI. However, when apoptosis is measured over time, cells can often be tracked from V450 Annexin V and PI negative (viable, or no measurable apoptosis), to V450 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to V450 Annexin V and PI 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 V450 Annexin V and PI positive, in and of itself, reveals less information about the process by which the cells underwent their demise.   This reagent is conjugated to BD Horizon™ V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon™ V450 can be used in place of Pacific Blue™ conjugates.

N/A Apoptosis is a normal physiological process that occurs during embryonic development and tissue homeostasis maintenance. The apoptotic program is characterized by certain morphological features, including plasma membrane symmetry and detachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of the plasma membrane is one of the earliest features of apoptosis. In apoptotic cells, phosphatidylserine (PS), a phospholipid membrane component, 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™ V450. 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, V450 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. V450 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 V450 Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (PI negative, V450 Annexin V positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. For example, cells that are considered viable are both V450 Annexin V and PI negative, cells that are in early apoptosis are V450 Annexin V positive and PI negative, and cells that are in late apoptosis or already dead are both V450 Annexin V and PI positive. This assay does not distinguish between cells that have undergone the apoptotic versus necrotic pathway because dead cells will be stained with both V450 Annexin V and PI. However, when apoptosis is measured over time, cells can often be tracked from V450 Annexin V and PI negative (viable, or no measurable apoptosis), to V450 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to V450 Annexin V and PI 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 V450 Annexin V and PI positive, in and of itself, reveals less information about the process by which the cells underwent their demise. This reagent is conjugated to BD Horizon™ V450, which has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser Ex max of 406 nm and has an Em Max at 450 nm. Conjugates with BD Horizon™ V450 can be used in place of Pacific Blue™ conjugates.

克隆 N/A (RUO)

Flow cytometry (Routinely Tested)

5 µl

Annexin V

Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.

研发参考(12) 1. 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). 2. Bossy-Wetzel E and Green D. Detection of apoptosis by Annexin V labeling. 2000; 322:15-18. (Biology). 3. 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). 4. 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). 5. 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). 6. 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). 7. O'Brien MC, Bolton WE. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry. 1995; 19(3):243-255. (Biology). 8. 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). 9. Schmid I, Krall WJ, Uittenbogaart CH, Braun J, Giorgi JV. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry. 1992; 13(2):204-208. (Biology). 10. Van Engeland, Nieland LJW, Ramaekers FCS, Schutte B, and Reutelingsperger CPM. . Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998; 31:1-9. (Biology). 11. 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). 12. 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).