《细胞健康检测》PPT课件.ppt

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 only a 15 minute fluorescent, non-lytic assay. Faster than AlamarBlue or MTT, MTS, XTT,Important to

3、 use appropriate filter sets,100 + 20,CellTiter-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

4、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 Cyto

5、toxicity Assay CytoTox-ONE Homogeneous 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 Fluorescence56

6、0Ex/590Em,10 minutes,Add 50l Stop Solution,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,Li

7、ght,AAF-Rhodamine110,AAF + Rhodamine 110 (485Ex/520Em),“dead cell” Protease released from cells withcompromised 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

8、 cytotoxicity assay,Sensitive to small 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

9、 Assay CytoTox-Glo,-Glo!,Fluor,Color,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 perm

10、eable, gets cleaved inside. Lcp rapidly degrades outside cell. “Dead cell” Protease (dcp) released from cells withcompromised membranes cleaves substrate correlates to cytotoxicity. Dcp substrate not cell permeable.,Luciferase + ATP + luciferin,Light,dcp,dcp,AAF-Rhodamine110,AAF + Rhodamine 110,GF-A

11、FC,GF + AFC,lcp,lcp,dcp,Glo,Fluor,MutiTox-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 chemis