流式细胞仪检测细胞凋亡操作流程 Annexin V 检测细胞凋亡.docx

Annexin V 检测细胞凋亡实验原理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S）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NA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+）mmH tic* rtrprcinnfaroH vf thrV 0AF岬 iff 应产皆h弘严”phgphs百difijirr取tr fPS P ra- jrtzNjJtK-iai'fiJ Fh* i町M 刖 Fkrp怙手 0吕 ftc - Irr fhr pnrwotf e/匚止忆 XriHi-*xrjj： V ka左曲high注津血上岁怛曾 PS. Ju nnfiximf 卩ofil* PS that hnrm『HpDHdT h ihf实验用品1. 一次性 12x75mm Falcon试管。

2. PBS缓冲液：含0.1%NaN3,过滤后2-8oC保存3. 微量加样器和加样头4. Annexin V Binding Buffer 缓冲液(Cat. No. 66121E )：浓度为 10x,使用时，用稀释为1x浓度的应用液5. Annexin V试剂与核酸染料：Annexin V核酸染料Ann exin V-Biotin (Cat. No. 65872X)Streptavidin-FITC(Cat. No. 13024D) Annexin V-FITC(Cat. No. 65874X)Annexin V-PE(Cat. No. 65875X)PI (Cat. No. 66211E)或7-AAD(Cat. No. 34321X)PI(Cat. No. 66211E)7-AAD(Cat. No. 34321X)6. FACS流式细胞仪：上样检测7. CELLQuest软件：获取和分析试验数据8. Annexin V检测对照管：Annexin VA-阴性对照B-补偿1C-补偿2BiotinSAv-FITCAnnexin V-Biotin 和 SAv-FITCPI和SAv-FITCPE未染色细胞Annexin V-PE7-AADFITC未染色细胞Annexin V-FITCPI操作步骤1. 取 Falcon 试管，按标本顺序编好阴性对照管和标本管号。

2 使用冷的PBS缓冲液洗细胞两次，再用1x Binding Buffer缓冲液制成1x106 细胞/ml的悬液3 Falcon试管中加入100山细胞悬液4 按以下体积加入Ann exin V与核酸染料：管号名称荧光标记Ann exin V核酸染料：1阴性对照--2单阳1AV-FITC-3单阳2-PI4样本AV-FITCPI5轻轻混匀，室温(20°C ~25OC)避光处放置15分钟6 *使用Ann exin V-Biot in试剂进行检测时：• 1xBi nding Buffer缓冲液洗细胞一次，去上清• 1xBi nding Buffer缓冲液100卩I溶解SAv-FITC试剂0.5旳，加入到细胞管中• 轻轻混匀•加入5山PI,室温(20-25°C)避光处放置15分钟7各试验管中分别加入1xB in di ng Buffer缓冲液400卩l8. 1 小时内上流式细胞仪测定结果结果分析：以下4个样本分别是阴性对照、Annexin V单阳管、PI单阳管和试验管1 1.003:|、•:蠢--亠—I100 101 102 103 104FL1-Height100 101 102 103 104FL1-Height4.662100 101 102 103 104Annexin1.004100 101 102 103 104AnnexinUJnsaQ瓷BmAbLsE卜ig“rc 12. 1 low (ytfimrtrii- of Apcptotlc Cvlh lAin^ Anncxin V'-FI I L.Jurkat T cells were left untceat^d or we« treated for 4 hr with an Shuman Fas n)Abr ,kmt DX2 (Cdt. No. 3345OI5)unci Piottin G. CdH wtrp incubated with Anncxin 门匚 in a buffer cum al Ding PI and ansiy?ed by Aljw uytfinwtTy. (A) Untreated cetls 叽忙 priirmn)y Aiincxin V4ITC and V\ negative ilu^r Left quadrjnt, pjnei mdi- cating that the cells were viable and not undergoing npopt曲乩(B) After treatment with DX2 and Pmtein G, a ^nifk^ni nuiitbvr of 山曉 3 re Annex in V^IIC' ^sitive □nd PI nefldiivt* (Jowr ri^hi quatlrani. pa nd I 儿 jndicatt^ th/i th* 讥恤 arc In un t arly MJffenf 叫m叩怛朋 and jrt stHI viahle In adtliLicin, ,i pupuhtioii yf tells has 卩rm grwsed to a later stage of apopto&is and are skiiiud with both Pl and Annexin V, indicating that the cells are no lender viable.Annexin V Blocking待测细胞与未标记的重组Ann exin V预孵育，然后进行实验，是Ann exin V 细胞凋亡检测的质量控制。

原理是预先阻断Ann exin V-FITC的结合位点，这样 可以证明Ann exin V-FITC凋亡分析的特异性1. 使用冷的PBS缓冲液洗细胞两次，再用1xB in di ng Buffer缓冲液制成1x106 细胞/ml的悬液2. 在5ml的培养管中加入100卩l细胞悬液（约1x105个细胞）3. 加入5-15旳纯化的重组Ann exin V注意，不同的细胞系，以及凋亡的不同 阶段，其Ann exin V位点饱和所需要的纯化的重组Ann exin V含量不同某些 情况下，为达到最好效果，可以减少细胞数量，0.5x105个细胞加5-15旳纯 化的重组Ann exin V4. 轻轻混匀，室温反应15分钟5. 加入5山的Ann exin V-FITC或/和PI,轻轻混匀，室温避光处放置15分钟6. 各试验管中分别加入1xB inding Buffer缓冲液400山7. 1 小时内上流式细胞仪测定结果8. 结果分析：下图A、B为Anti-Fas抗体诱导3小时后的Jurkat T细胞，图C、D为 未处理的细胞；图A、C为Ann exin V-FITC染色的凋亡检测结果，图B、D 为 Annexin V Blocking 后的检测结果。

Treal ed with Human Fbb mAb(3 hr>-3+ Purtlied Annexin VUntroati&dUntreatedTreated withHuman Fa«mAb [3 hr)Annexin V^FITCI igtire 11. Flow Cytometric Analysis of Annexin V Staining and Blocking. Jurkat r cells were induced to undergo apoptosis by treatment with anti-Fas antibody clone DX2 (Catr Nu, 334SOD) and Protein G for 3 hr (A, B)r Other cultures were left Linlrciitcd (C#Q}. Cells were incubated with Anncxiri V-FJTC alone (AFC) or with rcccmbtnant Annexin V to block Anncxln V binding $iU3i prior to addihg Annexln V-FITC (Br D). After a 3 hr ireatment with DXZt a population of cells was Annexln V-FITC positive (A, M2 gate). Annedn V-FLI'C staining was blocked when cells were first incubfited with 卩uritied miHiibirhint Annexin V ilii. As expected, the cell pop- ulatirm that v^as nut Lrcuted with DX2 were primarily Ancivxin V m盼tNt (C)r rhe small number of Annex in V positive cells in ihc untreated population Hkely represents a basaJ levtl of apuptosS. Thi茂 w\is blocktxi w]icn Lulls w^m lint iiuukMted with purified rccun]bi[idi]l Aun^xin V (l>|, Iht- M 1 uml M2 gates demarcutc Annexin V-F1TC negative and positive populations, respectively.PharMingen推荐Annexin V细胞凋亡检测试剂盒规格货号Annexin V-FITC Apoptosis Detection Kit I:Part A： Annexin V-FITC(100 tests)PI (2ml)lxBinding Buffer 缓冲液(50ml)100tests6693KKAnnexin V-FITC Apoptosis Detection Kit II (Annexin V Blocking):Part A： Annexin V-FITC(100 tests)PI (2ml)1xBinding Buffer 缓冲液(50ml)Part B： Purified Recom。