盛兆生物 細(xì)胞健康檢測(cè)

細(xì)胞健康檢測(cè)2012.5.11王春紅編輯課件內(nèi)容ADME檢測(cè)細(xì)胞凋亡細(xì)胞活力檢測(cè)細(xì)胞毒性檢測(cè)谷胱甘肽測(cè)定線粒體功能檢測(cè)編輯課件ADME:毒藥物動(dòng)力學(xué)編輯課件對(duì)外源化學(xué)物ADME過(guò)程的研究意義可用來(lái)確定與毒性發(fā)生有關(guān)的靶器官或組織的暴露特征。可為在其他的毒性研究的劑量選擇提供有用數(shù)據(jù)。有助于闡明兩種或兩種以上外源化學(xué)物聯(lián)合毒作用的機(jī)制?？赏ㄟ^(guò)改變外源化學(xué)物ADME過(guò)程，以預(yù)防和治療外源化學(xué)物中毒。編輯課件代謝（轉(zhuǎn)化）類型

可分兩類:第一類包括氧化、還原及水解過(guò)程；第二類為結(jié)合過(guò)程，第一類轉(zhuǎn)化產(chǎn)物再經(jīng)與體內(nèi)某些代謝物結(jié)合，產(chǎn)物一般水溶性加大，利于排泄。（1）第一階段反應(yīng)（第一類型）：氧化、還原及水解等。氧化，如醇氧化、醛氧化、單胺氧化、氧化脫氫及N-氧化等；還原，如硝基還原成氨基（-NH2）。（2）第二階段反應(yīng)（第二類型）：即結(jié)合反應(yīng)，使藥失效，隨尿排出。含羥基、羧基、胺基的化合物與葡萄糖醛酸結(jié)合成酯、醚、酰胺化合物；硫酸可與酚類藥物及酚性類固醇結(jié)合成硫酸酯；N-甲轉(zhuǎn)移酶使伯胺、腫胺及叔胺甲基化，以S-腺苷甲硫氨酸作為甲基供應(yīng)體；磺胺類及芳香族氨基等在乙酰輔酶A參與下乙?；?。編輯課件ADME&Metabolism產(chǎn)品P450-Glo?Assays&ScreeningSystemsGSH-GloAssayUGT-Glo?Assays&ScreeningSystemsMAO-Glo?AssayPgp-Glo?AssaySystem編輯課件編輯課件編輯課件編輯課件UGT活性檢測(cè)哺乳類動(dòng)物最重要的結(jié)合反應(yīng)

藥物結(jié)構(gòu)中的功能基團(tuán)結(jié)合生成葡萄糖醛酸GA的結(jié)合物

UDPGA（尿核苷二磷酸葡萄糖醛酸）：GA的活性供體編輯課件編輯課件Pgp-Glo?AssaySystems

Pgp,也被稱為MDR1和ABCB1,是一種170kDa的完整的質(zhì)膜蛋白，其生物學(xué)功能為依賴ATP的藥物泵，在多藥抗藥性和某些不良藥物相互作用中發(fā)揮重要作用。Pgp-Glo?試劑盒檢測(cè)化合物對(duì)膜制備物中重組人類Pgp活性的影響。該試劑盒建立在依賴于ATP的螢火蟲螢光素酶反應(yīng)上，該反應(yīng)可產(chǎn)生光信號(hào)。ATP先與Pgp共同孵育，然后PgpATP酶反應(yīng)被終止,未被消耗的ATP由螢光素產(chǎn)生的發(fā)光信號(hào)而被檢測(cè)出來(lái)。Pgp依賴的發(fā)光信號(hào)的降低反映了Pgp對(duì)ATP的消耗，因此信號(hào)減少得越多，Pgp的活力就越高。相應(yīng)地，若待測(cè)樣品中含有可刺激PgpATP酶的化合物，這個(gè)反應(yīng)產(chǎn)生的發(fā)光信號(hào)會(huì)顯著低于未經(jīng)處理的對(duì)照組。編輯課件細(xì)胞凋亡（apoptosis/(programmedcelldeath,PCD)

細(xì)胞凋亡是指為維持內(nèi)環(huán)境穩(wěn)定，由基因控制的細(xì)胞自主的有序的死亡。細(xì)胞凋亡與細(xì)胞壞死不同，細(xì)胞凋亡不是一件被動(dòng)的過(guò)程，而是主動(dòng)過(guò)程，它涉及一系列基因的激活、表達(dá)以及調(diào)控等的作用，它并不是病理?xiàng)l件下，自體損傷的一種現(xiàn)象，而是為更好地適應(yīng)生存環(huán)境而主動(dòng)爭(zhēng)取的一種死亡過(guò)程。編輯課件Pre-Caspase3ToolsApaf-1Cytccaspase

8caspase

3caspase

9proteincleavage

(e.g.,PARP)DNACleavageFas/TNFReceptorFADDstaurosporineantitumoragentsCaspase-Glo2Caspase-Glo8Z-VAD-FMKXXCaspase-Glo9Caspase-Glo6編輯課件Caspase-3ToolsApaf-1Cytccaspase

8caspase

3caspase

9DNACleavageFas/TNFReceptorFADDstaurosporineantitumoragentsCaspase-Glo3/7Apo-OneCaspase-3/7CaspACEFluorimetricCaspACEColorimetricFITC-VAD-FMKAnti-ACTIVECaspase-3pAbAc-DEVD-CHO

inhibitorproteincleavage

(e.g.,PARP)X編輯課件PostCaspase-3Toolscaspase

3DNACleavageFas/TNFReceptorFADDstaurosporineantitumoragentsproteincleavage

(e.g.,PARP)Apaf-1Cytccaspase

8caspase

9Anti-PARPp85

FragmentpAbDeadEndColorimetricDeadEndFluorimetric編輯課件CaspaseAssaysCaspACE?AssaySystem,ColorimetricCaspACE?AssaySystem,FluorimetricApo-One?HomogeneousCaspase-3/7AssayCaspase-Glo2AssayCaspase-Glo3/7AssayCaspase-Glo6AssayCaspase-Glo8AssayCaspase-Glo9Assay-Glo!FluorColor編輯課件CaspACEAssaySystemsAc-DEVD-AMCAc-DEVD+AMCfluorometricAc-DEVD-pNAAc-DEVD+pNAcolorimetricCaspase1/3編輯課件Apo-OneHomogeneousCaspase-3/7AssayZ-DEVD-R110-DEVD-ZZ-DEVD

+Caspase3/7R110編輯課件ConsensusTetrapeptides

VDVAD:Caspase-2

DEVD:Caspase-3/7VEID:Caspase-6LETD:Caspase-8LEHD:Caspase-9UsingBioluminescencetoMonitorCaspaseActivityAAAAAAAA編輯課件100+100編輯課件細(xì)胞活力在細(xì)胞群體中總有一些因各種原因而死亡的細(xì)胞，總細(xì)胞中活細(xì)胞所占的百分比叫做細(xì)胞活力.編輯課件Cell-Titer產(chǎn)品CellTiter96Non-RadioactiveCellProliferationAssay(MTT)CellTiter96AQueousNon-RadioactiveCellProliferationAssay(MTS)CellTiter96AQueousOneSolutionCellProliferationAssay(MTS)CellTiter-BlueCellViabilityAssayCellTiter-FluorCellViabilityAssayCellTiter-GloLuminescentCellViabilityAssay

ColorFluor-Glo!編輯課件CellTiter96?1-4hr37°CAddCellTiter96Reagent(MTS/PMS)ReadAbsorbance490nmCellsarenotlysedsoifcolordevelopmentisnottoyourliking,putbackinincubator.MTSMTSformazan(soluble)PESreduced

PESNADHNAD+PEStransportsreducingequivalentsfromtheinteriorofthecelltotheexterior.Deadcellshavelostreducingability.編輯課件Onesolution/Twosolution100+20編輯課件AppearanceofCellTiter96AssayPlateDecreasingCellNumber編輯課件CellTiter-Blue?CellViabilityAssay:Resazurinfluorescentassay

1-4hr37°CAddCellTiter-Blue?ReagentReadFluorescence560Ex/590EmCat.#G3580,G3581,G3582Multiplexingcapability,purestresazurinCellsarenotlysedsoifcolordevelopmentisnottoyourliking,putbackinincubator.Mayneedoptimization.ResazurinResorufinResazurinisreducedbymetabolicallyactivecells.Deadcellscannotreducethecompound.ReducingEnzymesYoucanreadtheabsorbancebutsensitivitysuffers.

~100cellsvs.>3000cells編輯課件100+20編輯課件AppearanceofCellTiterBlueAssayPlateDecreasingCellNumber(570-600nm)編輯課件CellTiter-Fluor?CellViabilityAssay:live-cellproteasemeasuredcpdcp“Deadcell”ProteasereleasedfromcellswithcompromisedmembranesdoesnotcontributetosignalGF-AFCGF+AFClcplcp“LiveCell”Protease(lcp)withinintactcellscleaves

cellpermeableGF-AFC.LCPenzymedegraded

outsidecell,doesnotcontributetosignal.Add100μlof

GF-AFCdilutedinAssayBufferReadFluorescence

Live:400Ex/505Em0.5-3hoursCat.#G6080,G6081,andG6082Multiplexwithreporterassaysandotherluminescentandfluorescentassays;onlya15minutefluorescent,non-lyticassay.FasterthanAlamarBlueorMTT,MTS,XTTImportanttouseappropriatefiltersets編輯課件100+20編輯課件CellTiter-Glo10MinutesAddCellTiter-Glo?ReagentReadLuminescenceSensitivetoasfewas10cellsATPATPATPATPATPATPATPATPATPATPLuciferinOxyluciferin+LIGHTUltra-Glo?LuciferaseADPATPisrapidlydegradedindeadcellsanddoesnotcontributetosignal編輯課件編輯課件Review方法原理產(chǎn)品目錄號(hào)規(guī)格價(jià)格(/rxn)比色法MTSbyNADHCellTiter96?AQueousNon-RadioactiveCellProliferationAssayG5421G5430G544010005000500001873(1.87/rxn)6182(1.23/rxn)48537(0.97/rxn)CellTiter96?AQueousOneSolutionCellProliferationAssayG3582G3580G358120010005000623(3.1/rxn)2,242(2.24/rxn)7503(1.5/rxn)熒光法ResazurinbyNADHCellTiterBlue?AssayG8080G8081G808220ml100ml1000ml858(0.858/rxn)2445(0.489/rxn)21133(0.422/rxn)GF-AFCbyLivecellproteaseCellTiter-FlourAssayG6080G6081G608210ml5X10ml2X50ml961(9.61/rxn)3974(7.94/rxn)6105(6.1/rxn)發(fā)光法ATPCellTiter-Glo?LuminescentCellViabilityAssayG7570G7571G7572G757310ml10X10ml100ml10X100ml726(7.26/rxn)3369(3.36/rxn)2856(2.85/rxn)22108(2.2rxn)編輯課件細(xì)胞毒性細(xì)胞毒性是由細(xì)胞或者化學(xué)物質(zhì)引起的單純的細(xì)胞殺傷事件，不依賴于凋亡或壞死的細(xì)胞死亡機(jī)理。細(xì)胞毒性檢測(cè)主要是根據(jù)細(xì)胞膜通透性發(fā)生改變來(lái)進(jìn)行的檢測(cè)，常用以下幾種方法：MTT、XTT法：利用線粒體內(nèi)部酶的活性，可以將特定的四唑鹽類進(jìn)行轉(zhuǎn)化，然后通過(guò)酶標(biāo)儀進(jìn)行檢測(cè)LDH的方法：通過(guò)檢測(cè)細(xì)胞培養(yǎng)上清中LDH的酶活性，來(lái)檢測(cè)細(xì)胞毒性其它酶方法：如檢測(cè)上清中堿性磷酸酶、酸性磷酸酶的活性等編輯課件編輯課件細(xì)胞毒性細(xì)胞毒性是由細(xì)胞或者化學(xué)物質(zhì)引起的單純的細(xì)胞殺傷事件，不依賴于凋亡或壞死的細(xì)胞死亡機(jī)理。編輯課件細(xì)胞毒性產(chǎn)品CytoTox96Non-RadioactiveCytotoxicityAssayCytoTox-ONEHomogeneousMembraneIntegrityAssayCytoTox-FluorCytotoxicityAssayCytoTox-Glo編輯課件CytoTox-ONE?AssayHomogeneousMembraneIntegrityAssay:FluorescentLDHAssayLDHLDHResazurinResorufinLactate+NAD+Pyruvate+NADHDiaphoraseAdd100μlof

CytoTox-One?ReagentReadFluorescence

560Ex/590Em10minutesAdd50μlStopSolutionAlsoworksifyouremoveportionofconditionedmediatonewplateandperformassay.Allowsmultiplexingonremainingcells.編輯課件100+100+50編輯課件CytoTox-GloandCytoTox-FluorAssaysdcpdcpAAF-aminoluciferin(notcellpermeable)luciferinATP+Ultra-Glo?LuciferaseLightdcpdcpAAF-Rhodamine110AAF+Rhodamine110(485Ex/520Em)“deadcell”Proteasereleasedfromcellswith

compromisedmembranescleavessubstrateCanbemultiplexedwithLuminescentCaspaseassays.CytoToxGloCytoToxFluor編輯課件CytoToxFluor100+100編輯課件CytoToxGlo100+50+50編輯課件CytoTox-Glo-themostsensitivecytotoxicityassaySensitivetosmallchangeinviabilityNofluorescenceinterferenceStablebiomarkerandluciferaseenzymeallowlargerassaywindow編輯課件Whatarethedifferences?CytoTox96Non-RadioactiveCytotoxicityAssayCytoTox-ONEHomogeneousMembraneIntegrityAssayCytoTox-FluorCytotoxicityAssayCytoTox-Glo-Glo!FluorColor編輯課件Review方法原理產(chǎn)品目錄號(hào)規(guī)格價(jià)格(/rxn)熒光法ResazurinbyLDHCytoTox-ONEAssayG7890G7891G8082200-8001000-40001000-40001034(5-1.29/rxn)3204(3.2-0.8/rxn)3051(3-0.7/rxn)AAF-R110byDeadcellproteaseCytoTox-FlourAssayG9200G9201G920210ml5X10ml2X50ml1759(17.59/rxn)4815(9.63/rxn)8747(8.7/rxn)發(fā)光法AAF-aminoluciferinbyDeadcellproteaseCytoTox-GloAssayG9290G9291G929210ml5X10ml2X50ml1009(10.09/rxn)3974(7.94/rxn)5814(5.814/rxn)編輯課件MultiPlexProductsMultiTox-FluorMultiplexCytotoxicityAssayMultiTox-GloMultiplexCytotoxicityAssayApoTox-GloTriplexAssayApoLive-Glo?MultiplexAssay編輯課件MultiTox-GloandMultiTox-FluorLiveandDeadCellMeasuresLivecellsubstrate(lcp)iscellpermeable,getscleavedinside.Lcprap