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4、the property of Becton, Dickinson and Company. 2004 BD559763 (Was: 6900KK) rev. 004page 1 of 3BD Pharmingen Technical Data SheetANNEXIN V-PE APOPTOSIS DETECTION KIT IPRODUCT INFORMATIONCatalog Number:559763Components:51-65875X:Contents:Annexin V-PE100 tests; buffered in 50 mM Tris (pH 8.0) with 80 m
5、M NaCl, 0.2%BSA, 1 mM EDTA, 0.09% (w/v) sodium azide.51-68981E:Contents:7-AAD (7-Amino-actinomycin D)2.0 ml in PBS and 0.09% sodium azide and FBS Wash Buffer.51-66121E:Contents:Annexin V Binding Buffer, 10X Concentrate50 ml solutionBACKGROUNDApoptosis is a normal physiologic process which occurs dur
6、ing 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 andattachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. Loss of plasma
7、 membrane is one of theearliest features. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leafletof the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependentphospholipid-bin
8、ding protein that has a high affinity for PS, and binds to cells with exposed PS (reviewed in 1). Annexin V may beconjugated to fluorochromes such as Phycoerythrin (PE). This format retains its high affinity for PS and thus serves as a sensitive probefor flow cytometric analysis of cells that are un
9、dergoing apoptosis.2-5Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V-PE staining can identify apoptosis at an earlier stage thanassays based on nuclear changes such as DNA fragmentation. Annexin V-PE staining precedes the loss of membrane integrity whichaccompanies
10、the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V-PE istypically used in conjunction with a vital dye such as 7-Amino-actinomycin (7-AAD, Cat. No. 68981E) to allow the investigator toidentify early apoptotic cells (Annexin V-PE
11、positive, 7-AAD negative).2-5 For example, cells that are viable are Annexin V-PE and 7-AADnegative; cells that are in early apoptosis are Annexin V-PE positive and 7-AAD negative; and cells that are in late apoptosis or alreadydead are both Annexin V-PE and 7-AAD positive.2-5 This assay does not di
12、stinguish, per se, between cells that have already undergoneapoptotic death and those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with bothAnnexin-PE and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annex
13、in V-PE and7-AAD negative (viable, or no measurable apoptosis), to Annexin V-PE positive and 7-AAD negative (early apoptosis, membraneintegrity is present) and finally to Annexin V-PE and 7-AAD positive (end stage apoptosis and death). The movement of cells throughthese three stages suggests apoptos
14、is. In contrast, a single observation indicating that cells are both Annexin V-PE and 7-AAD positive,in of itself, reveals less information about the process by which the cells underwent their demise.SPECIFICITY AND PREPARATIONA n ne xi n V -PE i s a s e n si ti v e pr obe for i d en ti fyi n g apop
15、toti c ce l l s.2-5 It bi n ds to n e g ati v e ly ch ar g e d ph os ph ol ipi d sur fa ce s (K d of 5 x 1 0 -2)6w i th a hi g h e r s peci fi ci ty for ph os ph a ti d y l se r i ne (PS) th an m ost othe r ph os phol i pid s . De fi n ed cal ci um a n d s a l t con cen tr ati on s a r e r e q uir e
16、 d for A n n exi n V-PE bi n d in g a s d e s cr i be d i n th e An n e xin V -PE Stai n i ng Pr otocol. Puri fi e d r e com bi na n t An n e xin V wa s con j uga te d to PEun de r opti m um cond i ti on s . An n e xin V -PE i s r outi n e l y te s te d usi n g pr i m ar y cel l s or ce ll l in e s in d uced to un d e rg o an a poptotic d e ath .NOTE: Methods for utilizing Annexin V binding on adherent cells (i.e., monolayer) have been described by van Engeland et al8 and Casciola-Rosenetal7. Ho
