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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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1楼2010-12-25 15:15:38

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三磷酸腺苷

铁杆木虫 (职业作家)

chzhbin(金币+30):呵呵是老师留的作业做期末考评的哈哈我也是给别人翻译的感谢 2010-12-25 17:41:16

凋亡检测。Caspase活性使用Apo-ONE Caspase-3/7-assay (Promega)试剂盒检测，根据说明书操作。简言之，于96孔板中每孔种50000个细胞，贴壁24h，后用分离的化合物孵育24h。随后，加入50xxx（看不清单位）的Apo-ONE Caspase-3/7试剂，在37℃中用Perkin-Elmer Wallac VICTOR 1420多标记分析仪（激发波长：485nm，发射波长：535nm）测定3h内荧光值的动态增长。
针对细胞凋亡中核碎裂的研究，首先将35mm皿中的细胞培养48h，后用不同浓度的类黄酮化合物孵育24h。随后在不换液的条件下，用xxx（数量和单位）的Hoechst 33342染细胞核15min，后封上盖玻片。用Zeiss Axiolab荧光显微镜 (激发波长: 365 nm, 发射波长: 420 nm)放大400倍拍摄带有固缩和碎裂核的细胞。

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