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1、细胞健康检测,2012.5.11 王春红,内容,ADME检测 细胞凋亡 细胞活力检测 细胞毒性检测 谷胱甘肽测定 线粒体功能检测,ADME:毒药物动力学,对外源化学物ADME过程的研究意义,可用来确定与毒性发生有关的靶器官或组织的暴露特征。 可为在其他的毒性研究的剂量选择提供有用数据。 有助于阐明两种或两种以上外源化学物联合毒作用的机制。 可通过改变外源化学物ADME过程，以预防和治疗外源化学物中毒。,代谢（转化）类型,可分两类:第一类包括氧化、还原及水解过程；第二类为结合过程，第一类转化产物再经与体内某些代谢物结合，产物一般水溶性加大，利于排泄。 （1）第一阶段反应（第一类型）：氧化、还原及
2、水解等。氧化，如醇氧化、醛氧化、单胺氧化、氧化脱氢及N-氧化等；还原，如硝基还原成氨基（-NH2）。 （2）第二阶段反应（第二类型）：即结合反应，使药失效，随尿排出。含羟基、羧基、胺基的化合物与葡萄糖醛酸结合成酯、醚、酰胺化合物；硫酸可与酚类药物及酚性类固醇结合成硫酸酯；N-甲转移酶使伯胺、肿胺及叔胺甲基化，以S-腺苷甲硫氨酸作为甲基供应体；磺胺类及芳香族氨基等在乙酰辅酶A参与下乙酰化。,ADME & Metabolism产品,P450-Glo Assays & Screening Systems GSH-Glo Assay UGT-Glo Assays & Screening Systems
3、 MAO-Glo Assay Pgp-Glo Assay System,UGT活性检测,哺乳类动物最重要的结合反应 药物结构中的功能基团结合生成葡萄糖醛酸GA的结合物 UDPGA（尿核苷二磷酸葡萄糖醛酸）：GA的活性供体,Pgp-Glo Assay Systems,Pgp, 也被称为MDR1 和ABCB1, 是一种 170kDa 的完整的质膜蛋白，其生物学功能为依赖ATP 的药物泵，在多药抗药性和某些不良药物相互作用中发挥重要作用。 Pgp-Glo 试剂盒检测化合物对膜制备物中重组人类Pgp 活性的影响。该试剂盒建立在依赖于ATP 的萤火虫萤光素酶反应上，该反应可产生光信号。ATP 先与Pgp
4、 共同孵育，然后Pgp ATP 酶反应被终止, 未被消耗的 ATP 由萤光素产生的发光信号而被检测出来。Pgp依赖的发光信号的降低反映了Pgp 对ATP 的消耗，因此信号减少得越多，Pgp 的活力就越高。相应地，若待测样品中含有可刺激Pgp ATP 酶的化合物，这个反应产生的发光信号会显著低于未经处理的对照组。,细胞凋亡（apoptosis /(programmed cell death,PCD),细胞凋亡是指为维持内环境稳定，由基因控制的细胞自主的有序的死亡。细胞凋亡与细胞坏死不同，细胞凋亡不是一件被动的过程，而是主动过程，它涉及一系列基因的激活、表达以及调控等的作用，它并不是病理条件下，自
5、体损伤的一种现象，而是为更好地适应生存环境而主动争取的一种死亡过程。,Pre-Caspase 3 Tools,Apaf-1,Cyt c,protein cleavage (e.g., PARP),DNA Cleavage,Fas/TNF Receptor,FADD,staurosporine,antitumor agents,Caspase-Glo 2 Caspase-Glo 8,Z-VAD-FMK,X,X,Caspase-Glo 9 Caspase-Glo 6,Caspase-3 Tools,Apaf-1,Cyt c,DNA Cleavage,Fas/TNF Receptor,FADD,st
6、aurosporine,antitumor agents,Caspase-Glo 3/7 Apo-One Caspase-3/7 CaspACE Fluorimetric CaspACE Colorimetric,FITC-VAD-FMK Anti-ACTIVE Caspase-3 pAb,Ac-DEVD-CHO inhibitor,protein cleavage (e.g., PARP),X,Post Caspase-3 Tools,DNA Cleavage,Fas/TNF Receptor,FADD,staurosporine,antitumor agents,protein cleav
7、age (e.g., PARP),Apaf-1,Cyt c,Anti-PARP p85 Fragment pAb,DeadEnd Colorimetric DeadEnd Fluorimetric,Caspase Assays,CaspACE Assay System, Colorimetric CaspACE Assay System, Fluorimetric Apo-One Homogeneous Caspase-3/7 Assay Caspase-Glo 2 Assay Caspase-Glo 3/7 Assay Caspase-Glo 6 Assay Caspase-Glo 8 As
8、say Caspase-Glo 9 Assay,-Glo!,Fluor,Color,CaspACE Assay Systems,Ac-DEVD-AMC Ac-DEVD + AMC fluorometric,Ac-DEVD-pNA Ac-DEVD + pNA colorimetric,Caspase 1/3,Apo-One Homogeneous Caspase-3/7 Assay,Z-DEVD-R110-DEVD-Z,Z-DEVD +,Caspase 3/7,R110,Consensus Tetrapeptides VDVAD: Caspase-2 DEVD: Caspase-3/7 VEID
9、: Caspase-6 LETD: Caspase-8 LEHD: Caspase-9,Using Bioluminescence to Monitor Caspase Activity,AAAA,AAAA,100 + 100,细胞活力,在细胞群体中总有一些因各种原因而死亡的细胞，总细胞中活细胞所占的百分比叫做细胞活力.,Cell-Titer 产品,CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (M
10、TS) CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) CellTiter-Blue Cell Viability Assay CellTiter-Fluor Cell Viability Assay CellTiter-Glo Luminescent Cell Viability Assay,Color,Fluor,-Glo!,CellTiter 96,1-4hr 37C,Add CellTiter 96Reagent (MTS/PMS),Read Absorbance 490nm,Cells are not
11、lysed so if color development is not to your liking, put back in incubator.,MTS,MTS formazan (soluble),PES,reduced PES,NADH,NAD+,PES transports reducing equivalents from the interior of the cell to the exterior. Dead cells have lost reducing ability.,One solution/Two solution,100 + 20,Appearance of
12、CellTiter 96 Assay Plate,Decreasing Cell Number,CellTiter-Blue Cell Viability Assay: Resazurin fluorescent assay,1-4hr 37C,Add CellTiter-Blue Reagent,Read Fluorescence 560Ex/590Em,Cat. # G3580, G3581, G3582 Multiplexing capability, purest resazurin,Cells are not lysed so if color development is not
13、to your liking, put back in incubator. May need optimization.,You can read the absorbance but sensitivity suffers. 100 cells vs. 3000 cells,100 + 20,Appearance of CellTiter Blue Assay Plate,Decreasing Cell Number (570-600nm),CellTiter-Fluor Cell Viability Assay: live-cell protease measure,dcp,dcp,“D
14、ead cell” Protease released from cells with compromised membranes does not contribute to signal,GF-AFC,GF + AFC,lcp,lcp,“Live Cell” Protease (lcp) within intact cells cleaves cell permeable GF-AFC. LCP enzyme degraded outside cell, does not contribute to signal.,Add 100l of GF-AFC diluted in Assay B
15、uffer,Read Fluorescence Live: 400Ex/505Em,0.5-3 hours,Cat. # G6080, G6081, and G6082 Multiplex with reporter assays and other luminescent and fluorescent assays; only a 15 minute fluorescent, non-lytic assay. Faster than AlamarBlue or MTT, MTS, XTT,Important to use appropriate filter sets,100 + 20,C
16、ellTiter-Glo,10 Minutes,Add CellTiter-Glo Reagent,Read Luminescence,Sensitive to as few as 10 cells,ATP,ATP,ATP,ATP,ATP,ATP,ATP,ATP,ATP,ATP,Luciferin,Oxyluciferin + LIGHT,Ultra-Glo Luciferase,ADP,ATP is rapidly degraded in dead cells and does not contribute to signal,Review,细胞毒性,细胞毒性是由细胞或者化学物质引起的单纯的
17、细胞杀伤事件，不依赖于凋亡或坏死的细胞死亡机理。 细胞毒性检测主要是根据细胞膜通透性发生改变来进行的检测，常用以下几种方法： MTT、XTT法：利用线粒体内部酶的活性，可以将特定的四唑盐类进行转化，然后通过酶标仪进行检测 LDH的方法：通过检测细胞培养上清中LDH的酶活性，来检测细胞毒性 其它酶方法：如检测上清中碱性磷酸酶、酸性磷酸酶的活性等,细胞毒性,细胞毒性是由细胞或者化学物质引起的单纯的细胞杀伤事件，不依赖于凋亡或坏死的细胞死亡机理。,细胞毒性产品,CytoTox 96 Non-Radioactive Cytotoxicity Assay CytoTox-ONE Homogeneous
18、Membrane Integrity Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo,CytoTox-ONE Assay Homogeneous Membrane Integrity Assay: Fluorescent LDH Assay,Resazurin,Resorufin,Lactate + NAD+,Pyruvate + NADH,Diaphorase,Add 100l of CytoTox-One Reagent,Read Fluorescence 560Ex/590Em,10 minutes,Add 50l Stop Solu
19、tion,Also works if you remove portion of conditioned media to new plate and perform assay. Allows multiplexing on remaining cells.,100 + 100 + 50,CytoTox-Glo and CytoTox-Fluor Assays,AAF-aminoluciferin (not cell permeable),luciferin,ATP + Ultra-Glo Luciferase,Light,AAF-Rhodamine110,AAF + Rhodamine 1
20、10 (485Ex/520Em),“dead cell” Protease released from cells with compromised membranes cleaves substrate,Can be multiplexed with Luminescent Caspase assays.,CytoTox Glo,CytoTox Fluor,CytoTox Fluor,100 + 100,CytoTox Glo,100 + 50 + 50,CytoTox-Glo - the most sensitive cytotoxicity assay,Sensitive to smal
21、l change in viability No fluorescence interference Stable biomarker and luciferase enzyme allow larger assay window,What are the differences?,CytoTox 96 Non-Radioactive Cytotoxicity Assay CytoTox-ONE Homogeneous Membrane Integrity Assay CytoTox-Fluor Cytotoxicity Assay CytoTox-Glo,-Glo!,Fluor,Color,
22、Review,MultiPlex Products,MultiTox-Fluor Multiplex Cytotoxicity Assay MultiTox-Glo Multiplex Cytotoxicity Assay ApoTox-Glo Triplex Assay ApoLive-Glo Multiplex Assay,MultiTox-Glo and MultiTox-Fluor Live and Dead Cell Measures,Live cell substrate (lcp) is cell permeable, gets cleaved inside. Lcp rapid
23、ly degrades outside cell. “Dead cell” Protease (dcp) released from cells with compromised membranes cleaves substrate correlates to cytotoxicity. Dcp substrate not cell permeable.,Luciferase + ATP + luciferin,Light,dcp,dcp,AAF-Rhodamine110,AAF + Rhodamine 110,GF-AFC,GF + AFC,lcp,lcp,dcp,Glo,Fluor,Mu
24、tiTox-Fluor,100 + 100,MutiTox-Glo,100 + 50 + 50,ApoTox-Glo Triplex Assay chemistry,Technical basis of the assay Viability/Cytotoxicity Assay (MultiTox-Fluor) Caspase-Glo 3/7 Assay,Promega Confidential Information. For Internal Use Only.,ApoTox-Glo,100 + 20 + 100,Triplex: Viability/Toxicity and Apoptosis,ApoLive-Glo Mutiplex Assay chemistry,Technical basis of the assay Viability Assay (Celltiter-Fluor) Caspase-Glo 3/7 Assay,Promega Confidential Information. For Internal Use Only.,ApoLive-Glo,100 + 20 + 100,谢谢！,