Annexin V Binding Apoptosis Assay Kit 膜联蛋白V细胞凋亡检测试剂盒中文说明书

检测细胞凋亡的三种流式方法细胞凋亡（apoptosis）是细胞自主性死亡的过程，是正常细胞发育和组织调控的重要途径。

为了研究细胞凋亡的机制和调控，科学家们发展出了许多方法来检测和定量细胞凋亡的发生。

其中流式细胞术（flow cytometry）是最常用的方法之一，根据不同的细胞特征和所需信息，可以有多种方法用于检测细胞凋亡。

下面将介绍三种常用的流式细胞术方法。

1. 荧光标记的Annexin V/PI双染法：Annexin V是一种细胞外结合蛋白，具有高度特异性结合细胞凋亡的特点。

当细胞凋亡发生时，磷脂酰丝氨酸（PS）从细胞内翻转至细胞外，而Annexin V能够结合在PS上。

Propidium Iodide（PI）能够穿过损伤的细胞膜，结合到细胞核内的DNA分子上。

通过将Annexin V标记为绿色荧光染料，将PI标记为红色荧光染料，通过流式细胞术定量测量荧光信号的强度，可以区分出未凋亡细胞（Annexin V和PI双阴性），早期凋亡细胞（Annexin V阳性，PI阴性），晚期凋亡和坏死细胞（Annexin V和PI双阳性）等不同状态的细胞。

2.荧光标记的DNA碎片染色法：3. 荧光标记的Caspase活性检测法：Caspases是细胞凋亡过程中的关键酶，它们在给定的信号下被激活并参与调节细胞凋亡的执行阶段。

通过使用荧光标记的Caspase底物，如FLICA系列（Fluorochrome Inhibitor of Caspases）、CaspGLOW系列（Caspase-Glo Kits），可以测量细胞中Caspase的活性。

这些底物在被Caspase酶剪切后会释放出荧光信号，通过流式细胞技术可以测量荧光信号的强度。

由于Caspase活性与细胞凋亡过程密切相关，因此可以利用这些荧光标记底物来定量测量细胞凋亡的发生。

总结起来，流式细胞术是一种非常有效的方法用于检测细胞凋亡的发生。

Annexin V/PI双染法可以定量测量不同状态的细胞，DNA碎片染色法可以测量细胞凋亡的程度，荧光标记的Caspase活性检测法可以测量细胞中Caspase的活性。

碧云天生物技术/Beyotime Biotechnology订货热线: 400-1683301或800-8283301订货e-mail:******************技术咨询: *****************网址: 碧云天网站 微信公众号活细胞Caspase-3活性与Annexin V细胞凋亡检测试剂盒产品编号产品名称包装C1077S 活细胞Caspase-3活性与Annexin V细胞凋亡检测试剂盒20次C1077M 活细胞Caspase-3活性与Annexin V细胞凋亡检测试剂盒50次产品简介：碧云天生产的活细胞Caspase-3活性与Annexin V细胞凋亡检测试剂盒(Caspase-3 Activity and Apoptosis Detection Kit for Live Cell)是基于新型的具有细胞膜通透性的Caspase-3/7绿色荧光底物GreenNuc™Caspase-3 Substrate联合细胞凋亡红色荧光探针Annexin V-mCherry来检测培养细胞中Caspase-3活性和细胞凋亡的红绿荧光双染试剂盒，可用于实时监测活细胞中Caspase-3活性和凋亡情况。

本试剂盒适用于流式细胞仪、荧光显微镜或其它荧光检测系统进行检测。

细胞凋亡(Apoptosis)是生物体发育等生命过程中普遍存在的、由基因决定的细胞主动有序的死亡方式。

当细胞遇到内、外环境因子刺激时，启动基因调控的自杀保护措施，去除体内非必需细胞或即将发生特化的细胞。

在这一过程中，细胞脱落离体或裂解为若干凋亡小体，并迅速被巨噬细胞或邻近细胞清除，这是一种由基因控制、高度有序的细胞自主死亡，包含一系列信号事件组成的通路。

细胞凋亡失调与多种疾病有关，例如阿尔茨海默病(Alzheimer’s disease)和癌症等。

Caspase (cysteine-dependent aspartate-specific proteases)的全称为天冬氨酸特异性的半胱氨酸蛋白水解酶，存在于蛋白质中，主要利用半胱氨酸(cysteine)侧链选择性地高效切割含有天冬氨酸(aspartate)的多肽底物，在介导细胞凋亡(apoptosis)过程中起重要作用，并参与细胞的炎症、生长和分化等过程。

请在使用前仔细阅读说明书Annexin V,FITC 凋亡检测试剂盒(100次)产品名称货号规格储存条件运输条件Annexin V,FITC结合物AD01-10 100次×1 0-5℃，避光(切勿冻存) 室温PI Solution AD02-05 50次×2 0-5℃，避光(切勿冻存) 室温10×Annexin V Binding Buffer AD03-05 50次×2 0-5℃，避光(切勿冻存) 室温*注：规格中的每“次”是以细胞浓度1×106 cells/ml计算产品描述细胞凋亡是指为维持有机体内环境稳定，由基因控制的细胞自主的有序的死亡。

正常情况下任何细胞在形成过程中发生的异常都会通过凋亡消除。

例如体内的癌细胞增长为肿瘤的过程会受细胞凋亡的引导而被抑制。

然而在抑癌基因p53出现问题时，凋亡就不会诱导发生，从而导致癌细胞的不断增长。

细胞凋亡可以通过细胞形态的变化或生物化学的变化来检测。

目前常用的指标有caspase活性变化、DNA碎片、磷脂酰丝氨酸的外翻等。

Annexin V染色的细胞可以用于检测细胞凋亡早期的细胞膜变化。

在细胞凋亡早期，膜磷脂酰丝氨酸由脂膜内侧翻向外侧。

Annexin V 是一种分子量为35~36kD的Ca2+依赖性磷脂结合蛋白，与磷脂酰丝氨酸有高度亲和力，可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜特异性结合，因此Annexin V 被作为检测细胞早期凋亡的灵敏指标之一。

用绿色荧光FITC标记的Annexin V 通过流式细胞仪或荧光显微镜可以检测到细胞凋亡的发生。

碘化丙啶(Propidium Iodide, PI)是一种核酸染料，PI只能透过凋亡晚期和死细胞的细胞膜，因此Annexin V和PI结合使用，可以区分凋亡早晚期的细胞及死细胞。

所需的设备和材料-合适量程的移液枪-样品和诱导剂-细胞培养用6，12，24，96孔板-PBS、去离子水-流式细胞仪或荧光显微镜。

PH0536|Annexin V-PE细胞凋亡检测试剂盒（Annexin V-PE apoptosis detection kit）货号：PH0536规格：PH0536-20|20T存储：4℃保存，半年有效。

AnnexinV-PE需避光保存。

◆产品组分试剂组成规格保存Annexin V-PE100uL4℃Annexin V-PE结合液12mL4℃，避光说明书1份/◆产品简介Annexin V-PE细胞凋亡检测试剂盒(Annexin V-PE Apoptosis Detection Kit)是用PE(Phycoerythrin)标记的重组人Annexin V来检测细胞凋亡时出现在细胞膜表面的磷酯酰丝氨酸的一种细胞凋亡检测试剂盒。

可以使用流式细胞仪、荧光显微镜或其它荧光检测设备进行检测。

本试剂盒检测的是红色荧光，因此在待检测的细胞已经表达GFP等绿色荧光蛋白的情况下，特别适合使用本试剂盒检测细胞凋亡。

PE可以被495nm、545或564nm的激发光所激发，发出峰值为575nm的荧光。

(参考图1)Annexin是一类广泛分布于真核细胞细胞浆内钙离子依赖的磷酯结合蛋白，参与细胞内的信号转导。

但仅Annexin V被报道可以调控一些PKC的活性。

Annexin V选择性结合磷酯酰丝氨酸(phosphatidylserine，简称PS)。

磷酯酰丝氨酸主要分布在细胞膜内侧，即与细胞浆相邻的一侧。

在细胞发生凋亡的早期，不同类型的细胞都会把磷酯酰丝氨酸外翻到细胞表面，即细胞膜外侧。

磷酯酰丝氨酸暴露到细胞表面后会促进凝血和炎症反应。

而Annexin V和外翻到细胞表面的磷酯酰丝氨酸结合后可以阻断磷酯酰丝氨酸的促凝血和促炎症反应活性。

用带有红色荧光的荧光探针PE标记的Annexin V，即Annexin V-PE，就可以用流式细胞仪或荧光显微镜非常简单而直接地检测到磷酯酰丝氨酸的外翻这一细胞凋亡的重要特征。

正常细胞不会被Annexin V-PE所染色，凋亡细胞和坏死细胞都会被Annexin V-PE所染色。

凯基Annexin V-EGFP细胞凋亡检测试剂盒（Annexin V-EGFP Apoptosis Detection Kit）Cat number：KGA For Research Use OnlyStore at4℃ for one yearExpire date：一、 试剂盒说明在正常细胞中，磷脂酰丝氨酸（PS）只分布在细胞膜脂质双层的内侧，而在细胞凋亡早期，细胞膜中的磷脂酰丝氨酸（PS）由脂膜内侧翻向外侧。

Annexin V是一种分子量为35~36kD的Ca2+依赖性磷脂结合蛋白，与磷脂酰丝氨酸有高度亲和力，故可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。

因此Annexin V被作为检测细胞早期凋亡的灵敏指标之一。

将Annexin V进行荧光素（EGFP、FITC）标记，以标记了的Annexin V作为荧光探针，利用荧光显微镜或流式细胞仪可检测细胞凋亡的发生。

与FITC的绿色荧光信号相比，EGFP的绿色荧光信号具有信号强，不易淬灭，稳定性高等优点，故本试剂盒采用EGFP作为荧光标记探针。

碘化丙啶（Propidium Iodide, PI）是一种核酸染料，它不能透过完整的细胞膜，但对凋亡中晚期的细胞和死细胞，PI能够透过细胞膜而使细胞核染红。

因此将Annexin V与PI匹配使用，就可以将处于不同凋亡时期的细胞区分开来。

本试剂盒可应用于培养细胞凋亡检测（不推荐用于检测组织样本）。

二、 试剂盒组份组份Cat: KGA10110 assays Cat: KGA10220 assaysCat: KGA10350 assaysCat: KGA104100 assays储存条件AnnexinV-EGFP 50μL 100μL 250μL 500μL 4℃避光Propidium Iodide 50μL 100μL 250μL 500μL 4℃避光Binding Buffer 5 mL 10.0 mL 25 mL 50 mL 4℃三、 试剂盒以外自备仪器和试剂流式细胞仪或荧光显微镜、低速离心机、微量移液器1.5m L Microtube、载玻片、盖玻片（荧光显微镜观察需用）、PBS、不含EDTA的胰酶消化液四、 使用注意事项1．微量试剂取用前请离心集液。

流式细胞仪检测细胞凋亡方法（AnnexinV法）实验概要Annexin V是一种检测细胞凋亡的试剂，本实验介绍了一个用流式细胞仪检测细胞凋亡的方法(Annexin V 法)。

实验原理Annexin V是检测细胞凋亡的灵敏指标之一。

它是一种磷脂结合蛋白，可以与早期凋亡细胞的胞膜结合，而细胞质膜的改变是细胞发生凋亡时最早的改变之一。

在细胞发生凋亡时，膜磷脂酰丝氨酸(PS)由质膜内侧翻向外侧。

Annexin V与磷脂酰丝氨酸有高度亲和力，因而与细胞外侧暴露的磷脂酰丝氨酸结合。

由于在发生凋亡时，磷脂酰丝氨酸外翻的发生早于细胞核的改变，因此，与DNA碎片检测比较，使用Annexin V可以更早地检测到凋亡细胞。

因为细胞坏死时也会发生磷脂酰丝氨酸外翻，所以Annexin V常与鉴定细胞死活的核酸染料(如PI或7-AAD)合并使用，来区分凋亡细胞(Annexin V /核酸染料-)与死亡细胞(Annexin V /核酸染料 )。

主要试剂1. PBS缓冲液：含0.1%NaN3，过滤后2-8°C保存。

2. Annexin V Binding Buffer缓冲液(Cat. No. 66121E)：浓度为10×，使用时，用稀释为1×浓度的应用液。

3. Annexin V试剂与核酸染料：Annexin V 核酸染料Annexin V-Biotin(Cat. No. 65872X) PI(Cat. No. 66211E)或Streptavidin-FITC(Cat. No. 13024D) 7-AAD(Cat. No. 34321X)Annexin V-FITC(Cat. No. 65874X) PI(Cat. No. 66211E) Annexin V-PE(Cat. No. 65875X) 7-AAD(Cat. No. 34321X)1. 一次性12×75mm Falcon试管。

BD Biosciences Pharmingen United States 877.232.8995Canada 888.259.0187Europe 32.53.720.211Japan 0120.8555.90Asia Pacific 65.6861.0633Latin America/Caribbean 55.11.5185.9995For country-specific contact information, visit /how_to_order/Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express BD 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 mM 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 Concentrate 50 ml solutionBACKGROUNDApoptosis 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 Ca 2+ dependent phospholipid-binding protein that has a high affinity for PS, and binds to cells with exposed PS (reviewed in 1). Annexin V may be conjugated to fluorochromes such as Phycoerythrin (PE). 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.2-5Since externalization of PS occurs in the earlier stages of apoptosis, Annexin V-PE staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. Annexin V-PE 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 Annexin V-PE is typically used in conjunction with a vital dye such as 7-Amino-actinomycin (7-AAD, Cat. No. 68981E) to allow the investigator to identify early apoptotic cells (Annexin V-PE positive, 7-AAD negative).2-5 For example, cells that are viable are Annexin V-PE and 7-AAD negative; cells that are in early apoptosis are Annexin V-PE positive and 7-AAD negative; and cells that are in late apoptosis or already dead are both Annexin V-PE and 7-AAD positive.2-5 This assay does not distinguish, per se, between cells that have already undergone apoptotic death and those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both Annexin-PE and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Annexin V-PE and 7-AAD negative (viable, or no measurable apoptosis), to Annexin V-PE positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Annexin V-PE 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 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 n e x i n V -PE i s a s e n s i t i v e pr o be for i d e n t i f yi n g apoptoti c ce l l s .2-5 It bi n d s to n e g a ti v e l y ch a r g e d ph o s p h o l i pi d sur f a c e s (K d of ~5 x 1 0 -2)6w i t h a hi g h e r s p eci f i c i t y for ph o s p h a t i d y l s e r i n e (PS) th a n m o st othe r ph o s p hol i p id s . De f i n e d cal c i u m a n d s a l t con c en t r a ti o n s a r e r e q u ir e d for A n n e xi n V-PE bi n d i n g a s d e s c r i b e d i n th e An n e x in V -PE Stai n i n g Pr o tocol. Puri f i e d r e c om b i n a n t An n e x in V wa s con j u ga t e d to PE un d e r opti m u m con d i t i o n s . An n e x in V -PE i s r o uti n e l y te s t e d usi n g pr i m a r y cel l s or ce l l l i n e s in d u ced to un d e r g o an a p optotic d e a th .NOTE: Methods for utilizing Annexin V binding on adherent cells (i.e., monolayer) have been described by van Engeland et al 8 and Casciola-Rosen etal 7. However, these methods are not routinely tested for Annexin V-PE. AnnexinV-FITC Microscopy kit (Cat.No. 550911) is recommended for detection of Annexin V binding to adherent cells.USAGE AND STORAGEA p pli c a t ion s in c l u d e flow cytom e tr y (5 µl /tes t ). Se e th e A n n e xi n V-PE Stai n i n g Pr o tocol for us a g e i n for m a t ion . Stor e A n n e xi n V-PE at 4 ° C .(p l e a se see n e x t p a g e )BD Biosciences Pharmingen United States 877.232.8995Canada 888.259.0187Europe 32.53.720.211Japan 0120.8555.90Asia Pacific 65.6861.0633Latin America/Caribbean 55.11.5185.9995For country-specific contact information, visit /how_to_order/Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express REFERENCES1.Raynal, P. and H.B. Pollard. 1994. Annexins: The problem of assessing the biological role for a gene family of multifunctional calcium and phospholipid-binding proteins. Biochemica et Biophysica Acta. 1197:63-93.2.Vermes, I., C. Haanen, H. Steffens-Nakken, and C. Reutelingsperger. 1995. A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Meth. 184:39-51.3.Martin, S.J., C.P. Reutelingsperger, A.J. McGahon, J.A. Rader, R.C. van Schie, D.M. LaFace, and D.R. Green. 1995. Early redistribution of plasmamembrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J.Exp. Med. 182:1545-1556.4.Koopman, G., C.P. Reutelingsperger, G.A. Kuijten, R.M. Keehnen, S.T. Pals, and M.H. van Oers. 1994. Annexin V for flow cytometric detection ofphosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415-1420.5. Homburg, C.H., M. de Haas, A.E. von dem Borne, A.J. Verhoeven, C.P. Reutelingsperger and D. Roos. 1995. Human neutrophils lose their surfaceFc γRIII and acquire Annexin V binding sites during apoptosis in vitro . Blood 85:532-540.6. Andree, H.A., C.P. Reutelingsperger, R. Hauptmann, H.C. Hemker, W.T. Hermens and G.M. Willems. 1990. Binding of vascular anticoagulant α(VAC α) to planar phospholipid-binding proteins. J. Biol. Chem. 265:4923-4928.7. Casciola-Rosen, L., A. Rosen, M. Petri, and M. Schlissel. 1996. Surface blebs on apoptotic cells are sites of enhanced procoagulantactivity:Implications for coagulation events and antigenic spread in systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 93:1624-1629.8. van Engeland, M., F.C. Ramaekers, B. Schutte, and C.P. Reutelingsperger. 1996. A novel assay to measure loss of plasma membrane asymmetryduring apoptosis of adherent cells in culture. Cytometry 24:131-139.ANNEXIN V -PE STAINING PROTOCOLAnnexin V -PE is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external environment.Annexin V is a Ca 2+-dependent phospholipid-binding protein that has a high affinity for PS, and is useful for identifying apoptotic cells with exposed PS. 7-Amino-actinomycin (7-AAD) is a standard flow cytometric viability probe and is used to distinguish viable from nonviable cells. Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. Cells that stain positive for Annexin V-PE and negative for 7-AAD are undergoing apoptosis. Cells that stain positive for both Annexin V-PE and 7-AAD are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both Annexin V-PE and 7-AAD are alive and not undergoing measurable apoptosis.Reagents1.Annexin V-PE (Cat. No. 51-65875X). Use 5 µl per test.2.7-AAD (Cat. No. 51-68981E). Use 5 µl per test. 7-AAD (7-Amino-actinomycin D) is a convenient, ready-to-use solution of the nucleicacid dye that can be used for the exclusion of nonviable cells in flow cytometric assays. 7-AAD fluorescence is detected in the far red range of the spectrum (650 nm long-pass filter).1,23. 1 0 X A n n e x i n V B i nd i n g B u f fe r . (Ca t . N o . 5 1 -66 1 2 1 E ). 0.1 M H e p e s /N a O H (pH 7 .4 ) 1 .4 M N a Cl , 25 m M Ca C l 2.3Th e s o luti o n w a s 0 .2 µm s t e r i l e fil t e r e d . For a w o r k i n g s o l u tion (1X ), di l u te 1 pa r t bi n d i n g buffe r to 9 par t s di s t i l l e d H 20. T h i s w i l l y i e l d a wor k i n g s o luti o n of 1 0 m M H e p e s /N a O H (pH 7 .4 ) 1 4 0 m M Na C l , 2 .5 mM Ca C l 2. Stor e both th e 1 0 X con c e n tr a t e a n d w o r k i n g sol u tion a t 2 - 8 ° C .Staining1.Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 106 cells/ml.2.Transfer 100 µl of the solution (1 x 105 cells) to a 5 ml culture tube.3.Add 5 µl of Annexin V -PE and 5 µl of 7-AAD.4.Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.5.Add 400 µl of 1X binding buffer to each tube. Analyze by flow cytometry within one hour.For Research Use Only. Not For Diagnostic or Therapeutic Use.Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD PharMingen will not be held responsible for patent infringement or other violations that may occur with the use of our products.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.Conditions: The information disclosed herein is not to be construed as a recommendation to use the above product in violation of any patents. BD Biosciences will not be held responsible for patent infringement or other violations that may occur with the use of our products. Purchase does not include or carry any right to resell or transfer this product either as a stand-alone product or as a component of another product. Any use of this product other than the permitted use without the express 10010110210310M1M2100Annexin V-PE7-A 10110210310410010110101010010110210310M1M2B T reated100Annexin V-PE7-A A D101102103104100101102103104Annexin V-PE: A tool for identifying cells that are undergoing apoptosis.T cells were left untreated (A) or treated for 4 hr with 4 µM Camptothecin (B). Cells were incubated with Annexin V -PE in a buffer containing 7-Amino-actinomycin (751-68981E) and analyzed by flow cytometry. Untreated primarily Annexin V -PE and 7-AAD negative, indicating that they were viable and SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRYThe following controls are used to set up compensation and quadrants 1.Unstained cells.2.Cells stained with Annexin V -PE alone (no 7-AAD).3.Cells stained with 7-AAD alone (no Annexin V-PE).Other Staining ControlsA cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with Annexin V-PE and with both Annexin V-PE 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 at least a minor percentage of cells that are positive for apoptosis (Annexin V-PE positive, 7-AAD negative or Annexin V-PE and 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 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 Annexin V-PE. 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 Annexin V-PE and 7-AAD.INDUCTION OF APOPTOSIS BY CAMPTOTHECINAnnexin V-PE。

膜联蛋白V的结合细胞凋亡检测试剂盒绿色荧光优化流式细胞仪Cat.No ：BS7127包装：1套一、描述膜联蛋白是一类钙依赖性磷脂结合蛋白。

它们大量存在于真核生物中，是参与信号转导的广泛的细胞质蛋白家族。

膜联蛋白V的优先结合伴侣是磷脂酰丝氨酸（PS），它通常保持在细胞膜的内表面（胞质一侧）。

在细胞凋亡时，PS转移到质膜的外表面。

细胞表面上的磷脂酰丝氨酸是细胞凋亡起始/中间阶段的一个通用指标，可以在观察形态变化之前被检测出。

我们的细胞仪表™检测试剂盒是通过检测磷脂酰丝氨酸（PS）的易位来监测细胞凋亡的。

该试剂盒采用我们专有的绿色荧光膜联蛋白V- iFluor 488 PS传感器，它能够特异性结合PS 。

染色具有光谱特性，几乎与更高光稳定性的FITC相同，从而使其方便用于配备光源和滤波器的共同荧光仪器。

该试剂盒提供了所有与流式细胞仪的应用优化的协议的必需成分。

二、试剂盒成分A组分：膜联蛋白V- iFluor 488 （100X原液）1瓶（200微升/瓶）B组份：缓冲液50毫升C 组分：100X碘化丙啶1瓶（100微升）三、保存4℃，避光四、步骤注意：对贴壁细胞的膜联蛋白V流式细胞术分析不是常规检测，因为在细胞脱落或收获时，可能会出现特定的膜损伤。

但是，此前已报道Casiola -罗森等人对贴壁细胞类型利用Annexin V的流式细胞仪的方法。

1、用试验化合物处理细胞（喜树碱处理的Jurkat细胞4-6小时）来诱导细胞凋亡。

2、离心细胞，得到1-5 × 105个细胞/管。

3、将细胞悬浮于200μL的缓冲液（B组分）中。

4、添加2μL膜联蛋白V- iFluor™ 488（A组分）到细胞中。

可选：加入2 μL 100X碘化丙啶（C组分）到坏死细胞中。

5、在室温下孵育30 〜60分钟，避光。

6、可选：加200-300μL缓冲液（B组分），以增加体积在细胞仪分析细胞（见步骤7）。

之前。

7、使用FL1通道显示膜联蛋白V- iFluor ™ 488的荧光强度（EX / EM =490/525nm），并使用FL2通道和碘化丙啶检测细胞活力。