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Determination of ACD. Caspase activity was measured using the Apo-ONE homogeneous Caspase-3/7-assay (Promega) according to the manufacturers protocol. Briefly, 50 000 cells/well were plated in 96-multiwell dishes, allowed to attach for 24 h, and treated with isolated compounds for 24 h. Then 50 µL of Apo-ONE Caspase-3/7-reagent was added, and the increase in fluorescence was measured for3hat 37 °C using a Perkin-Elmer Wallac VICTOR 1420 multilabel counter (excitation: 485 nm, emission: 535 nm). To investigate nuclear fragmentation as a further feature of apoptotic cell death, cells were grown in 35 mm cell culture dishes for 48 h and then incubated with different concentrations of the flavonoid compounds for 24 h. Then, without medium change, nuclei were stained with 100 µmol/L Hoechst 33342 for 15 min and sealed with a glass coverslip. Cells with condensed and fragmented nuclei were photographed at 400-fold magnification using a Zeiss Axiolab fluorescence microscope (excitation: 365 nm, emission: 420 nm). |
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鐵桿木蟲 (職業(yè)作家)
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鐵桿木蟲 (職業(yè)作家)
鐵桿木蟲 (職業(yè)作家)
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凋亡檢測。Caspase活性使用Apo-ONE Caspase-3/7-assay (Promega)試劑盒檢測,根據(jù)說明書操作。簡言之,于96孔板中每孔種50000個細(xì)胞,貼壁24h,后用分離的化合物孵育24h。隨后,加入50xxx(看不清單位)的Apo-ONE Caspase-3/7試劑,在37℃中用Perkin-Elmer Wallac VICTOR 1420多標(biāo)記分析儀(激發(fā)波長:485nm,發(fā)射波長:535nm)測定3h內(nèi)熒光值的動態(tài)增長。 針對細(xì)胞凋亡中核碎裂的研究,首先將35mm皿中的細(xì)胞培養(yǎng)48h,后用不同濃度的類黃酮化合物孵育24h。隨后在不換液的條件下,用xxx(數(shù)量和單位)的Hoechst 33342染細(xì)胞核15min,后封上蓋玻片。用Zeiss Axiolab熒光顯微鏡 (激發(fā)波長: 365 nm, 發(fā)射波長: 420 nm)放大400倍拍攝帶有固縮和碎裂核的細(xì)胞。 校友的份上…… |
鐵桿木蟲 (職業(yè)作家)
榮譽版主 (文壇精英)






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