201x盛兆生物 细胞健康检测 (2)

1、整理课件细胞健康检测王春红整理课件内容nADME检测n细胞凋亡n细胞活力检测n细胞毒性检测n谷胱甘肽测定n线粒体功能检测整理课件ADME:毒药物动力学整理课件对外源化学物ADME过程的研究意义n可用来确定与毒性发生有关的靶器官或组织的暴露特征。 n可为在其他的毒性研究的剂量选择提供有用数据。 n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。 n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。 整理课件代谢（转化）类型代谢（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。 n（1）第

2、一阶段反应（第一类型）：氧化、还原及水解等。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）。 n（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。 整理课件ADME & Metabolism产品产品P450-Glo Assays & Screening SystemsGSH-Glo Assay UGT-Glo Ass

3、ays & Screening SystemsMAO-Glo Assay Pgp-Glo Assay System 整理课件整理课件整理课件整理课件UGT活性检测活性检测n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的活性供体整理课件整理课件Pgp-Glo Assay SystemsnPgp, 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。 nPgp-Glo 试剂盒检测化合物对膜制备物中重组人类Pgp 活

4、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 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。整理课件细胞凋亡（apoptosis /(programmed cell death,PCD) n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

5、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。 整理课件Pre-Caspase 3 ToolsApaf-1Cyt ccaspase8caspase3caspase9protein cleavage(e.g., PARP)DNA CleavageFas/TNFReceptorFADDstaurosporineantitumoragentsCaspase-Glo 2Caspase-Glo 8Z-VAD-FMKXXCaspase-Glo 9Caspase-Glo 6整理课

6、件Caspase-3 ToolsApaf-1Cyt ccaspase8caspase3caspase9DNA CleavageFas/TNFReceptorFADDstaurosporineantitumoragentsCaspase-Glo 3/7Apo-One Caspase-3/7CaspACE FluorimetricCaspACE ColorimetricFITC-VAD-FMKAnti-ACTIVE Caspase-3 pAbAc-DEVD-CHOinhibitorprotein cleavage(e.g., PARP)X整理课件Post Caspase-3 Toolscaspas

7、e3DNA CleavageFas/TNFReceptorFADDstaurosporineantitumoragentsprotein cleavage(e.g., PARP)Apaf-1Cyt ccaspase8caspase9Anti-PARP p85Fragment pAbDeadEnd ColorimetricDeadEnd Fluorimetric整理课件Caspase AssaysCaspACE Assay System, ColorimetricCaspACE Assay System, FluorimetricApo-One Homogeneous Caspase-3/7 A

8、ssayCaspase-Glo 2 AssayCaspase-Glo 3/7 AssayCaspase-Glo 6 AssayCaspase-Glo 8 AssayCaspase-Glo 9 Assay -Glo!FluorColor整理课件CaspACE Assay SystemsAc-DEVD-AMC Ac-DEVD + AMC fluorometricAc-DEVD-pNA Ac-DEVD + pNA colorimetricCaspase 1/3整理课件Apo-One Homogeneous Caspase-3/7 AssayZ-DEVD-R110-DEVD-ZZ-DEVD +Casp

9、ase 3/7R110整理课件ConsensusTetrapeptides VDVAD: Caspase-2 DEVD: Caspase-3/7VEID: Caspase-6LETD: Caspase-8LEHD: Caspase-9Using Bioluminescence to Monitor Caspase ActivityAAAAAAAA整理课件100 + 100整理课件细胞活力n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. 整理课件Cell-Titer 产品nCellTiter 96 Non-Radioactive Cell Prolifera

10、tion Assay (MTT)nCellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)nCellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)nCellTiter-Blue Cell Viability AssaynCellTiter-Fluor Cell Viability AssaynCellTiter-Glo Luminescent Cell Viability Assay ColorFluor -Glo!整理课件CellTiter

11、961-4hr37CAdd CellTiter 96Reagent (MTS/PMS)ReadAbsorbance490nmCells are not lysed so if color development is not to your liking, put back in incubator.MTSMTSformazan(soluble)PESreducedPESNADHNAD+PES transports reducing equivalents from the interior of the cell to the exterior. Dead cells have lost r

12、educing ability.整理课件One solution/Two solution100 + 20整理课件Appearance of CellTiter 96 Assay PlateDecreasing Cell Number整理课件CellTiter-Blue Cell Viability Assay: Resazurin fluorescent assay 1-4hr37CAdd CellTiter-Blue ReagentReadFluorescence560Ex/590EmCat. # G3580, G3581, G3582Multiplexing capability, pu

13、rest resazurinCells are not lysed so if color development is not to your liking, put back in incubator. May need optimization.ResazurinResorufinResazurin is reduced by metabolically active cells. Dead cells cannot reduce the compound.Reducing EnzymesYou can read the absorbance but sensitivity suffer

14、s.100 cells vs. 3000 cells整理课件100 + 20整理课件Appearance of CellTiter Blue Assay PlateDecreasing Cell Number(570-600nm)整理课件CellTiter-Fluor Cell Viability Assay: live-cell protease measuredcpdcp“Dead cell” Proteasereleased from cells with compromised membranes does not contribute to signalGF-AFCGF + AFCl

15、cplcp“Live Cell” Protease (lcp)within intact cells cleavescell permeable GF-AFC. LCP enzyme degradedoutside cell, does not contribute to signal.Add 100l of GF-AFC diluted in Assay BufferRead FluorescenceLive: 400Ex/505Em0.5-3 hoursCat. # G6080, G6081, and G6082 Multiplex with reporter assays and oth

16、er luminescent and fluorescent assays; only a 15 minute fluorescent, non-lytic assay.Faster than AlamarBlue or MTT, MTS, XTTImportant to use appropriate filter sets整理课件100 + 20整理课件CellTiter-Glo10MinutesAdd CellTiter-Glo ReagentReadLuminescenceSensitive to as few as 10 cellsATPATPATPATPATPATPATPATPAT

17、PATPLuciferinOxyluciferin + LIGHTUltra-GloLuciferaseADPATP is rapidly degraded in dead cells and does not contribute to signal整理课件整理课件Review 方法方法原理原理产品产品目录号目录号规格规格价格价格(/rxn)比色法比色法MTS by NADHCellTiter 96 AQueous Non-Radioactive Cell Proliferation AssayG5421G5430G5440 1000 5000 500001873 (1.87/rxn)618

18、2 (1.23/rxn)48537 (0.97/rxn)CellTiter 96 AQueous One SolutionCell Proliferation AssayG3582G3580G3581200 1000 5000623(3.1/rxn) 2,242 (2.24/rxn)7503 (1.5/rxn)荧光法荧光法Resazurinby NADHCellTiter Blue AssayG8080G8081G808220ml100ml1000ml858 (0.858/rxn)2445 (0.489/rxn)21133 (0.422/rxn)GF-AFC by Live cell prot

19、ease CellTiter-Flour Assay G6080G6081G608210ml5X10ml2X50ml961(9.61/rxn)3974(7.94/rxn)6105(6.1/rxn)发光法发光法ATPCellTiter-Glo Luminescent Cell Viability AssayG7570G7571G7572G757310ml 10X10ml 100ml 10X100ml726 (7.26/rxn)3369 (3.36/rxn)2856 (2.85/rxn)22108 (2.2rxn)整理课件细胞毒性n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

20、或坏死的细胞死亡机理。 n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 整理课件整理课件细胞毒性n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。 整理课件细胞毒性产品CytoTox 96 Non-Radioactive Cytotoxicity AssayCytoTox-ONE Homogeneous Me

21、mbrane Integrity AssayCytoTox-Fluor Cytotoxicity Assay CytoTox-Glo 整理课件CytoTox-ONE Assay Homogeneous Membrane Integrity Assay: Fluorescent LDH AssayLDHLDHResazurinResorufinLactate + NAD+Pyruvate + NADHDiaphoraseAdd 100l of CytoTox-One ReagentRead Fluorescence560Ex/590Em10 minutesAdd 50lStop Solution

22、Also works if you remove portion of conditioned media to new plate and perform assay. Allows multiplexing on remaining cells.整理课件100 + 100 + 50整理课件CytoTox-Glo and CytoTox-Fluor AssaysdcpdcpAAF-aminoluciferin(not cell permeable)luciferinATP + Ultra-Glo LuciferaseLightdcpdcpAAF-Rhodamine110AAF + Rhoda

23、mine 110 (485Ex/520Em)“dead cell” Proteasereleased from cells withcompromised membranes cleaves substrateCan be multiplexed with Luminescent Caspase assays.CytoTox GloCytoTox Fluor整理课件CytoTox Fluor100 + 100整理课件CytoTox Glo100 + 50 + 50整理课件CytoTox-Glo - the most sensitive cytotoxicity assaynSensitive

24、to small change in viabilitynNo fluorescence interferencenStable biomarker and luciferase enzyme allow larger assay window整理课件What are the differences?CytoTox 96 Non-Radioactive Cytotoxicity AssayCytoTox-ONE Homogeneous Membrane Integrity AssayCytoTox-Fluor Cytotoxicity Assay CytoTox-Glo -Glo!FluorC

25、olor整理课件Review 方法方法原理原理产品产品目录号目录号规格规格价格价格(/rxn)荧光法荧光法Resazurinby LDHCytoTox-ONE AssayG7890G7891G8082200-8001000-40001000-40001034 (5-1.29/rxn)3204(3.2-0.8/rxn)3051 (3-0.7/rxn)AAF-R110 by Dead cell protease CytoTox-Flour Assay G9200G9201G920210ml5X10ml2X50ml1759(17.59/rxn)4815(9.63/rxn)8747(8.7/rxn)发

26、光法发光法AAF-amino luciferin by Dead cell proteaseCytoTox-Glo AssayG9290G9291G929210ml5X10ml2X50ml1009(10.09/rxn)3974(7.94/rxn)5814(5.814/rxn)整理课件MultiPlex ProductsMultiTox-Fluor Multiplex Cytotoxicity AssayMultiTox-Glo Multiplex Cytotoxicity AssayApoTox-Glo Triplex AssayApoLive-Glo Multiplex Assay 整理课件MultiTox-Glo and MultiTox-Fluor Live and Dead Cell MeasuresLive cell substrate (lcp) is cell permeable, gets cleaved ins