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Annexin V-FITC/PI 细胞凋亡检测试剂盒
产品货号:40302ES20
产品规格:20T
产品价格:¥480.00
类别:细胞凋亡与吞噬

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  •  产品信息

    产品名称

    货号

    规格

    价格(元)

    促销价(元)

    Annexin V-FITC/PI Apoptosis Detection Kit 

    Annexin V-FITC/PI 细胞凋亡检测试剂盒 

    40302ES20

    20 T

    480.00

    366.00

    40302ES50

    50 T

    960.00

    686.00

    40302ES60

    100 T

    1680.00

    986.00

     
    产品描述

    Annexin V-FITC/PI细胞凋亡检测试剂盒是用FITC标记的Annexin V作为探针,来检测细胞早期凋亡的发生

    其检测原理为:在正常的活细胞中,磷脂酰丝氨酸(phosphotidylserine,PS)位于细胞膜的内侧,但在早期凋亡的细胞中,PS 从细胞膜的内侧翻转到细胞膜的表面,暴露在细胞外环境中。Annexin-Ⅴ(膜联蛋白-V)是一种分子量为35-36 kDaCa2+ 依赖性磷脂结合蛋白,能与PS高亲和力结合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。

    另外,本试剂盒中还提供了碘化丙啶(Propidium Iodide,PI)用来区分存活的早期细胞和坏死或晚期凋亡细胞。PI是一种核酸染料,它不能透过正常细胞或早期凋亡细胞的完整的细胞膜,但可以透过凋亡晚期和坏死细胞的细胞膜而使细胞核染红。因此,将Annexin V与PI联合使用时,PI 则被排除在活细胞(Annexin V-/PI-)和早期凋亡细胞(Annexin V+/PI-)之外,而晚期凋亡细胞和坏死细胞同时被FITC 和PI 结合染色呈现双阳性(Annexin V+/PI+)。

    本试剂盒可用于流式细胞仪、荧光显微镜进行检测。


    产品组分

    编号

    组分

    产品编号/规格

    40302ES20(20T)

    40302ES5050T

    40302ES60100T

    40302-A

    Annexin V-FITC

    100 μL

    250 μL

    250 μL×2

    40302-B

    PI Staining Solution

    200 μL

    500 μL

    500 μL×2

    40302-C

    1×Binding Buffer

    10 mL

    25 mL

    25 mL×2


    运输与保存方法

    冰袋(wet ice)运输。-20℃避光长期保存,避免反复冻融。

    【注】:如果需要在短时间内多次重复使用,可以在4℃避光保存,半年有效。


    注意事项

    1)由于细胞凋亡是一个快速的过程,建议样品在染色后1小时之内进行分析。
    2) 对于贴壁细胞,消化是一个关键步骤。贴壁细胞诱导细胞凋亡时如有漂浮细胞,需收集漂浮细胞和贴壁细胞后合并染色。处理贴壁细胞时要小心操作,尽量避免人为的损伤。胰酶消化时间过短,细胞需要用力吹打才能脱落,容易造成细胞膜的损伤PI摄入过多消化时间过长,细胞膜同样易造成损伤,甚至会影响细胞膜上磷脂酰丝氨酸与Annexin V-FITC的结合。消化时将胰酶铺满孔板底后,轻摇使胰酶与细胞充分接触,然后倒掉大部分胰酶,利用剩余少量胰酶再消化一段时间,待细胞间空隙增大,瓶底呈花斑状即可终止。在消化液中尽量不用EDTAEDTA会影响Annexin V与PS的结合。
    3) 实验中如需要固定细胞,比如在检测凋亡的同时检测细胞周期,只能选用Annexin V-FITC,而不能选用Annexin V-EGFP,因为在固定过程中EGFP会变性导致丧失激发荧光的能力。固定前需要先将细胞与Annexin V-FITC进行孵育,并用Binding buffer洗掉未结合的Annexin V-FITC。因为固定过程中细胞通透性增加会产生细胞碎片,可以和Annexin V结合,对结果产生干扰。
    4)如果样品来源于血液,请务必除去血液中的血小板。因为血小板含有PS,能与Annexin V结合,从而干扰实验结果。可以使用含有EDTA的缓冲剂并在200 g离心洗去血小板。

    5)试剂在开盖前请短暂离心,将盖内壁上的液体甩至管底,避免开盖时液体洒落

    6)Annexin V-FITC和PI是光敏物质,在操作时请注意避光


    操作方法

    1.1 样品染色

    1)悬浮细胞300 g4离心5 min收集细胞。

    贴壁细胞:用不含EDTA的胰酶消化后,300 g4离心5 min收集细胞。胰酶消化时间不宜过长,以防引起假阳性。

    2)用预冷的PBS洗涤细胞2次,每次均需300 g4离心5 min。收集15×105细胞。

    3)吸弃PBS,加入100 μL 1×Binding Buffer重悬细胞。

    4)加入5 μL Annexin V-FITC10 μLPI Staining Solution,轻轻混匀。

    5)避光、室温反应10-15 min

    6)加入400 μL 1×Binding Buffer,混匀后放置于冰上,样品在1小时内用流式细胞仪或荧光显微镜检测。

    为了避免洗涤细胞时损失细胞,在吸液时可以用大的Tip头套上小的Tip头吸液。

    1.2 样品分析

    A.流式细胞仪分析:

    FITC最大激发波长为488 nm最大发射波长525 nm,FITC的绿色荧光在FL1通道检测PI-DNA复合物的最大激发波长为535 nm,最大发射波长为615 nm,PI的红色荧光在FL2或FL3通道检测。用CellQuest等软件进行分析,绘制双色散点图(two-color dot plot),FITC为横坐标,PI为纵坐标。典型的实验中,细胞可以分成三个亚群,活细胞仅有很低强度的背景荧光,早期凋亡细胞仅有较强的绿色荧光,晚期凋亡细胞有绿色和红色荧光双重染色。

    B.荧光显微镜分析:

    1滴一滴用Annexin V-FITC/PI双染的细胞悬液于载玻片上,并用盖玻片盖上细胞。

    对于贴壁细胞,可直接用盖玻片培养细胞并诱导细胞凋亡。

    2在荧光显微镜下用双色滤光片观察。Annexin V-FITC荧光信号呈绿色,PI荧光信号呈红色。

    数据展示

    数据来源:上海交通大学纳米生物工程研究所

    文章信息Cui, D., et al., Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying both Ligand and siRNA. Sci Rep, 2015. 5: p. 10726.

    细胞类型MGC803 cells;

    所用试剂:Yeasen,Cat:40302,Annexin V-FITC/PI Apoptosis Detection Kit

    image.png 

    “Fig5.Determination of cell death by flow cytometry of Annexin V-FITC/PI staining in MGC803 cells transfected with 25 nM FA-pRNA-3WJ-BRCAA1siRNA or FA-pRNA-3WJ-Scram-siRNA for 48 h.” 

    数据来源:中山大学;

    文章信息:Zhou, T., et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/beta-catenin signaling pathway in hippocampal neurons. Biochem Biophys Res Commun, 2017. 486(4): p. 1005-1013.

    细胞类型Hippocampal neurons

    所用试剂:Yeasen,Cat:40302 ,Annexin V-FITC/PI Apoptosis Detection Kit

    image.png 

    “Fig. 3. Wnt/b-catenin mediates the expression levels of apoptosis-related proteins and the protective effects of mild hypothermia against OGD/R-induced apoptosis。scatter diagram of PI/Annexin V gating from different groups. a. Control; b.OGD/R; c.OGD/R t MH(24 h); d.OGD/R t MH(24 h)tDkk1; e. OGD/R t Dkk1.”


    参考文献

    [1].Wei, W.J., et al., Propranolol sensitizes thyroid cancer cells to cytotoxic effect of vemurafenib. Oncol Rep, 2016. 36(3): p. 1576-84.

    [2].Wang, Q., et al., Fluorescent carbon dots as an efficient siRNA nanocarrier for its interference therapy in gastric cancer cells. J Nanobiotechnology, 2014. 12: p. 58.

    [3].Zhang, C., et al., Insights into the distinguishing stress-induced cytotoxicity of chiral gold nanoclusters and the relationship with GSTP1. Theranostics, 2015. 5(2): p. 134-49.

    [4].Tong, K., C. Xin and W. Chen, Isoimperatorin induces apoptosis of the SGC-7901 human gastric cancer cell line via the mitochondria-mediated pathway. Oncol Lett, 2017. 13(1): p. 518-524.

    [5].Tian, Y., et al., Tobacco Mosaic Virus-Based 1D Nanorod-Drug Carrier via the Integrin-Mediated Endocytosis Pathway. ACS Appl Mater Interfaces, 2016. 8(17): p. 10800-7.

    [6].Hou, W., et al., pH-Sensitive self-assembling nanoparticles for tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy. Nanoscale, 2016. 8(1): p. 104-16.

    [7].Hou, W., et al., pH-Sensitive self-assembling nanoparticles for tumor near-infrared fluorescence imaging and chemo-photodynamic combination therapy. Nanoscale, 2016. 8(1): p. 104-16.

    [8].Yu, H., et al., Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro. Exp Cell Res, 2016. 343(2): p. 218-22.

    [9].Li, J., et al., Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10  Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway. J Cancer, 2016. 7(13): p. 1877-1887.

    [10]. Yang, Y., et al., Human CIK Cells Loaded with Au Nanorods as a Theranostic Platform for Targeted Photoacoustic Imaging and Enhanced Immunotherapy and Photothermal Therapy. Nanoscale Res Lett, 2016. 11(1): p. 285.

    [11]. Zhuang, Y.T., et al., IL-6 induced lncRNA MALAT1 enhances TNF-alpha expression in LPS-induced septic cardiomyocytes via activation of SAA3. Eur Rev Med Pharmacol Sci, 2017. 21(2): p. 302-309.

    [12]. Wang, P., et al., Blood Plasma of Patients with Parkinson's Disease Increases Alpha-Synuclein Aggregation and Neurotoxicity. Parkinsons Dis, 2016. 2016: p. 7596482.

    [13]. Ge, G.F., et al., Baicalein attenuates vinorelbine-induced vascular endothelial cell injury and chemotherapeutic phlebitis in rabbits. Toxicol Appl Pharmacol, 2017. 318: p. 23-32.

    [14]. Ge, G.F., et al., Baicalein attenuates vinorelbine-induced vascular endothelial cell injury and chemotherapeutic phlebitis in rabbits. Toxicol Appl Pharmacol, 2017. 318: p. 23-32.

    [15]. Cai, G., et al., Galectin-3 induces ovarian cancer cell survival and chemoresistance via TLR4 signaling activation. Tumour Biol, 2016. 37(9): p. 11883-11891.

    [16]. Zhou, T., et al., Mild hypothermia protects against oxygen glucose deprivation/reoxygenation-induced apoptosis via the Wnt/beta-catenin signaling pathway in hippocampal neurons. Biochem Biophys Res Commun, 2017. 486(4): p. 1005-1013.

    [17]. Cui, D., et al., Regression of Gastric Cancer by Systemic Injection of RNA Nanoparticles Carrying  both Ligand and siRNA. Sci Rep, 2015. 5: p. 10726.

    [18]. Zhang, C., et al., A New Ligustrazine Derivative-Selective Cytotoxicity by Suppression of NF-kappaB/p65 and COX-2 Expression on Human Hepatoma Cells. Part 3. Int J Mol Sci, 2015. 16(7): p. 16401-13.

    [19]. Wu, Z., et al., LncRNA-N1LR Enhances Neuroprotection Against Ischemic Stroke Probably by Inhibiting p53 Phosphorylation. Mol Neurobiol, 2016.

    [20]. Ni, C., et al., A novel mutation in TRPV3 gene causes atypical familial Olmsted syndrome. Sci Rep, 2016. 6: p. 21815.

    [21]. Aipire, A., et al., Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine. Sci Rep, 2017. 7: p. 43796.

    [22]. Liu, H., et al., Targeting heat-shock protein 90 with ganetespib for molecularly targeted therapy of gastric cancer. Cell Death Dis, 2015. 6: p. e1595.


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