BD Pharmingen™ Cy™5 Annexin V

ANNEXIN V

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence 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 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 Cy™5. 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, Cy™5 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. Cy™5 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 Cy™5 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 (7-AAD negative, Cy™5 Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD.  For example, cells that are considered viable are both Cy™5 Annexin V and 7-AAD negative while cells that are in early apoptosis are Cy™5 Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Cy™5 Annexin V 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 Cy™5 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Cy™5 Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Cy™5 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Cy™5 Annexin V 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 Cy™5 Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence 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 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 Cy™5. 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, Cy™5 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. Cy™5 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 Cy™5 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 (7-AAD negative, Cy™5 Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD.  For example, cells that are considered viable are both Cy™5 Annexin V and 7-AAD negative while cells that are in early apoptosis are Cy™5 Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Cy™5 Annexin V 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 Cy™5 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Cy™5 Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Cy™5 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Cy™5 Annexin V 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 Cy™5 Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.

(RUO)

Flow cytometry (Routinely Tested)

5 µl

Annexin V

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

研发参考(7) 1. 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). 2. 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: Apoptosis). 3. 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). 4. 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: Apoptosis). 5. 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). 6. 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). 7. 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).