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上海信裕
1.当细胞铺满整个平皿(100mm)后,吸弃旧培养液,10mLPBS洗一遍
2.0.25%胰酶(预热20分钟左右)2ml消化,轻轻摇晃,MMP Substrate-3 MCA-Pro-Cha-Gly-Nva-His-Ala-DNP-NH2·TFA直至肉眼见单层细胞呈块状脱落(或在显微镜下观察细胞之间分离),立刻加5ml(DMEM+10%FBS)终止消化,轻柔吹打8-10次成单细胞。
3.离心1000rpm*5min,弃上清,加DMEM+10%FBS轻柔吹打。
4.将细胞悬液1:4传到100mm细胞培养皿中,每个皿加10ml(DMEM+10%FBS)培养液,正常情况下2-3天就可以长满,期间不用换液。
MMP Substrate-3 MCA-Pro-Cha-Gly-Nva-His-Ala-DNP-NH2·TFA的传代方法:首先在倒置显微镜下观察生长融合达80%,不需要长满,就准备传代。倒掉旧的培养基,再用已经高压灭菌过的PBS洗1-2次,我用的是25平方厘米的培养瓶,加入1毫升已经配制好的0.25%胰蛋白酶-0.02%EDTA(0.25%胰蛋白酶是用D-Hanks配制后先用滤纸过滤,再用一次性过滤器过滤除菌,MMP Substrate-3 MCA-Pro-Cha-Gly-Nva-His-Ala-DNP-NH2·TFA再分装成1ml的小包装,刚好每次用完一个,避免每次反复冻存,会使胰酶的活性下降),加入的量以刚好盖满瓶底为宜。放到倒置显微镜下观察变化,一旦发现细胞开始变园收缩,大约1-3分钟时间,必要时可以吹打帮助细胞分散,就立即加入培养基中和胰酶,如有EDTA最好要离心(800rpm,5min),弃上清夜,MMP Substrate-3 MCA-Pro-Cha-Gly-Nva-His-Ala-DNP-NH2·TFA再加入完全培养基吹匀,再分瓶,一般一瓶可以分2-3瓶。放入5%的CO2培养箱,37度培养24小时后观察。
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文献和实验Antibodies to MMP-Cleaved Aggrecan
of whether or not an MMP is active and acting on a particular substrate has been more difficult. If the primary sequence and unique cleavage sites within the substrate are known, one means of unambiguously identifying MMP activity is to use antibodies with a unique
themselves functioning as proMMP activators. Therefore, understanding the structural basis of MMP function, in particular substrate recognition and cleavage, MMP inhibition by TIMPs and synthetic inhibitors, and the domain:domain interactions that occur in the activation
often have overlapping substrate specificities: there are at least five commonly found collagenases, MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14 (MMP-18 is also a collagenase but has only been found so far in xenopus) which will all degrade triple helical collagen
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