《細胞健康檢測》PPT課件.ppt

1、細胞健康檢測,2012.5.11 王春紅,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,ADME:毒藥物動力學,對外源化學物ADME過程的研究意義,可用來確定與毒性發(fā)生有關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物聯(lián)合毒作用的機制。 可通過改變外源化學物ADME過程，以預防和治療外源化學物中毒。,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（第一類型）：氧化、還原及

2、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（第二類型）：即結(jié)合反應，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體；磺胺類及芳香族氨基等在乙酰輔酶A參與下乙酰化。,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,細胞毒性,細胞毒性是由細胞或者化學物質(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,細胞毒性,細胞毒性是由細胞或者化學物質(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。,細胞毒性產(chǎn)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