盛兆生物 細胞健康檢測

細胞健康檢測2012.5.11王春紅編輯課件內(nèi)容ADME檢測細胞凋亡細胞活力檢測細胞毒性檢測谷胱甘肽測定線粒體功能檢測編輯課件ADME:毒藥物動力學(xué)編輯課件對外源化學(xué)物ADME過程的研究意義可用來確定與毒性發(fā)生有關(guān)的靶器官或組織的暴露特征?？蔀樵谄渌亩拘匝芯康膭┝窟x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)合毒作用的機制?？赏ㄟ^改變外源化學(xué)物ADME過程，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、還原及水解過程；第二類為結(jié)合過程，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活性檢測哺乳類動物最重要的結(jié)合反應(yīng)

藥物結(jié)構(gòu)中的功能基團結(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?試劑盒檢測化合物對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。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)光信號而被檢測出來。Pgp依賴的發(fā)光信號的降低反映了Pgp對ATP的消耗，因此信號減少得越多，Pgp的活力就越高。相應(yīng)地，若待測樣品中含有可刺激PgpATP酶的化合物，這個反應(yīng)產(chǎn)生的發(fā)光信號會顯著低于未經(jīng)處理的對照組。編輯課件細胞凋亡（apoptosis/(programmedcelldeath,PCD)

細胞凋亡是指為維持內(nèi)環(huán)境穩(wěn)定，由基因控制的細胞自主的有序的死亡。細胞凋亡與細胞壞死不同，細胞凋亡不是一件被動的過程，而是主動過程，它涉及一系列基因的激活、表達以及調(diào)控等的作用，它并不是病理條件下，自體損傷的一種現(xiàn)象，而是為更好地適應(yīng)生存環(huán)境而主動爭取的一種死亡過程。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編輯課件細胞活力在細胞群體中總有一些因各種原因而死亡的細胞，總細胞中活細胞所占的百分比叫做細胞活力.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)品目錄號規(guī)格價格(/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)編輯課件細胞毒性細胞毒性是由細胞或者化學(xué)物質(zhì)引起的單純的細胞殺傷事件，不依賴于凋亡或壞死的細胞死亡機理。細胞毒性檢測主要是根據(jù)細胞膜通透性發(fā)生改變來進行的檢測，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行轉(zhuǎn)化，然后通過酶標(biāo)儀進行檢測LDH的方法：通過檢測細胞培養(yǎng)上清中LDH的酶活性，來檢測細胞毒性其它酶方法：如檢測上清中堿性磷酸酶、酸性磷酸酶的活性等編輯課件編輯課件細胞毒性細胞毒性是由細胞或者化學(xué)物質(zhì)引起的單純的細胞殺傷事件，不依賴于凋亡或壞死的細胞死亡機理。編輯課件細胞毒性產(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)品目錄號規(guī)格價格(/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