V500 Annexin V

V500

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™ V500. 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, V500 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. V500 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 V500 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, V500 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 V500 Annexin V and PI negative, cells that are in early apoptosis are V500 Annexin V positive and PI negative,  and cells that are in late apoptosis or already dead are both V500 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 V500 Annexin V and PI. However, when apoptosis is measured over time, cells can often be tracked from V500 Annexin V and PI negative (viable, or no measurable apoptosis), to V500 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to V500 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 V500 Annexin V and PI positive, in and of itself, reveals less information about the process by which the cells underwent their demise. Annexin V is conjugated to BD Horizon™ V500 that has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser with an Ex max of 415 nm and Em Max at 500 nm. BD Horizon V500 conjugates emit at a similar wavelength to Amcyan yet exhibit reduced spillover into the FITC channel. For more information on BD Horizon V500, visit bdbiosciences.com/colors. When compensating dyes in this spectral range (such as Horizon™ V500 and AmCyan), the most accurate compensation can be obtained using single stained cellular controls. Due to spectral differences between cells and beads in this channel, using BD™ CompBeads can result in spillover errors for V500 and AmCyan reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different V500 reagents (e.g. CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.

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™ V500. 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, V500 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. V500 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 V500 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, V500 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 V500 Annexin V and PI negative, cells that are in early apoptosis are V500 Annexin V positive and PI negative,  and cells that are in late apoptosis or already dead are both V500 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 V500 Annexin V and PI. However, when apoptosis is measured over time, cells can often be tracked from V500 Annexin V and PI negative (viable, or no measurable apoptosis), to V500 Annexin V positive and PI negative (early apoptosis, membrane integrity is present) and finally to V500 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 V500 Annexin V and PI positive, in and of itself, reveals less information about the process by which the cells underwent their demise. Annexin V is conjugated to BD Horizon™ V500 that has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. It is excited by the Violet laser with an Ex max of 415 nm and Em Max at 500 nm. BD Horizon V500 conjugates emit at a similar wavelength to Amcyan yet exhibit reduced spillover into the FITC channel. For more information on BD Horizon V500, visit bdbiosciences.com/colors. When compensating dyes in this spectral range (such as Horizon™ V500 and AmCyan), the most accurate compensation can be obtained using single stained cellular controls. Due to spectral differences between cells and beads in this channel, using BD™ CompBeads can result in spillover errors for V500 and AmCyan reagents. Therefore, the use of BD CompBeads or BD CompBeads Plus to determine spillover values for these reagents is not recommended. Different V500 reagents (e.g. CD4 vs. CD45) can have slightly different fluorescence spillover therefore, it may also be necessary to use clone specific compensation controls when using these reagents.

Flow cytometry (Routinely Tested)

5 µl

Human (QC Testing)

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

研发参考(8) 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. 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). 3. 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). 4. 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). 5. 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). 6. 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). 7. 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). 8. 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).