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胰岛素增敏活性筛选实验操作翻译
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胰岛素增敏活性筛选实验 用含10% FBS的高糖有酚红DMEM 培养基在六孔板中培养3T3-L1细胞，至汇合度达90%进行诱导分化，用含10% FBS，Ins, Dex, IBMX 的高糖有酚红DMEM 培养基诱导，四天后重新消化接种于24孔板，用含10% FBS的高糖有酚红DMEM 培养基培养两天。然后再用含10% FBS和较低浓度Ins较高浓度 Dex的高糖有酚红DMEM 培养基进行诱导胰岛素抵抗，四天后换用含待测样品的高糖无酚红DMEM培养基，同时设立空白对照(诱导分化后的正常细胞)和阴性对照(诱导分化后的胰岛素抗性细胞)不加样品。继续培养50h后取10ul 培养上清, 转到酶标板中，采用葡萄糖检测试剂盒检测葡萄糖水平，490nM测定OD值。实验时，每个样品设双复孔，重复两次。根据OD值计算增敏率，增敏率=[1-(OD样品-OD空白)/(OD阴性-OD空白)] ×100%，相对增敏率=样品增敏率/罗格列酮增敏率。 |
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wypward(金币+1):欢迎新虫，辛苦啦 2010-06-01 10:51:07
hanyan2004(金币+50, 翻译EPI+1): 2010-06-01 23:17:06
hanyan2004(金币+1): 2010-06-02 12:10:53
hanyan2004(金币+1):翻译得很好，谢谢你哈 2010-06-02 21:37:14
wypward(金币+1):欢迎新虫，辛苦啦 2010-06-01 10:51:07
hanyan2004(金币+50, 翻译EPI+1): 2010-06-01 23:17:06
hanyan2004(金币+1): 2010-06-02 12:10:53
hanyan2004(金币+1):翻译得很好，谢谢你哈 2010-06-02 21:37:14
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Screening test of Insulin-sensitizing activity 3T3-L1 cells were cultured in high glucose DMEM culture meduim with phenol red Containing 10% FBS in six-well plates. When confluence reached 90%, cells were induced to differentiate with a high sugar DMEM culture medium with phenol red containing 10% FBS, Ins, Dex, IBMX. After four days, cells were seeded in 24 well plates after re-digestion, cultured in high glucose DMEM culture medium with phenol red containing 10% FBS for two days. and then they were induced for insulin resistance using high glucose DMEM culture medium with phenol red containing 10% FBS, Ins in lower concentration and Dex in higher concentration. Four days later the cells were transferred to the high glucose DMEM medium without phenol red containing sample, while the control test (normal cells after induction of differentiation) and negative control test (insulin resistant cells after induced differentiation without sample) were established. 10ul supernatants were taken to ELISA plate after 50hs’ continuous culture to test the glucose level using glucose testing kit and OD values in 490nm. During the experiment, a double recovery was set for each sample hole and the same experiment was repeated twice. The increased sensitivity rate was calculated according to OD values, increased sensitivity rate = [1 - (OD sample-OD blank) / (OD negative-OD blank)] × 100%, the relative increased sensitivity rate = increased sensitivity of sample / increased sensitivity of rosiglitazone. |
2楼2010-06-01 10:22:34
