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MTT檢測技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/6/2閱讀:247
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關(guān)鍵詞:MTT檢測技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)
簡介:世界*品牌MTT檢測技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*,。
MTT檢測技術(shù)服務(wù)|實(shí)驗(yàn)技術(shù)服務(wù)——pCzn1質(zhì)粒+Arctic Express宿主菌低溫表達(dá)體系。
我們具備豐富的蛋白表達(dá)設(shè)計(jì)經(jīng)驗(yàn)及實(shí)驗(yàn)操作技巧,,承諾客戶不成功不收費(fèi),。在獲得重組蛋白產(chǎn)品的同時(shí),我們也會提供相應(yīng)的原始數(shù)據(jù)和原始圖片,不需要再額外花費(fèi)精力重復(fù)實(shí)驗(yàn),。另一方面,,我們所有的實(shí)驗(yàn)數(shù)據(jù)真實(shí)可信,,我們提供原始的表達(dá)菌株和克隆質(zhì)粒,,客戶可以依據(jù)我們的實(shí)驗(yàn)報(bào)告重復(fù)我們的實(shí)驗(yàn)結(jié)果,。
產(chǎn)品品牌:   http://www.。,。com/goodsid/fenleiyi/2868603/1.html    
測序?qū)嶒?yàn)流程:
MTT為黃色化合物,,是一種接受氫離子的染料,,在活細(xì)胞中作用于線粒體的呼吸鏈,MTT在琥珀酸脫氫酶和細(xì)胞色素C的作用下生成藍(lán)色的formazan結(jié)晶,,formazan結(jié)晶的生成量與活細(xì)胞數(shù)目成正比,,生成的formazan結(jié)晶可溶解在DMSO中,利用酶標(biāo)儀測定570 nm處的光密度OD值,,可以反映出活細(xì)胞數(shù)目和細(xì)胞活力,。死細(xì)胞中由于沒有琥珀酸脫氫酶,因此不能還原MTT生成formazan結(jié)晶,。
優(yōu)點(diǎn):經(jīng)濟(jì),、靈敏度高,可用于大規(guī)模的抗腫瘤藥物篩選,、細(xì)胞毒性試驗(yàn)以及腫瘤放射敏感性測定等,。
缺點(diǎn):由于MTT經(jīng)還原所產(chǎn)生的formazan結(jié)晶產(chǎn)物不溶于水,需有機(jī)溶劑溶解后才能檢測,,這不僅使工作量增加,也會對實(shí)驗(yàn)結(jié)果的準(zhǔn)確性產(chǎn)生影響,,而且有機(jī)溶劑對實(shí)驗(yàn)人員也有損害,。
①將對數(shù)生長期的細(xì)胞濃度調(diào)整為1~10×104細(xì)胞/ml,按103~104個(gè)細(xì)胞接種于96孔板,,每孔100 μl,;
②培養(yǎng)細(xì)胞24小時(shí)后,對其進(jìn)行不同處理,,每個(gè)處理設(shè)三個(gè)平行對照,;
③(MTT法)適當(dāng)培養(yǎng)時(shí)間后,取出細(xì)胞,,倒掉培養(yǎng)液,,每孔加入5 mg/ml新鮮配制的MTT溶液,溫育4小時(shí),,再次倒掉上清液,,每孔加入200 ml DMSO,搖勻后,,酶標(biāo)儀檢測吸光值,。
③(臺盼藍(lán)拒染法)適當(dāng)培養(yǎng)時(shí)間后,收集細(xì)胞,,經(jīng)臺盼藍(lán)染色,,在倒置顯微鏡下計(jì)數(shù)藍(lán)染及不染色細(xì)胞,計(jì)算細(xì)胞存活率,。
操作步驟:
1,、無菌條件下取新生一天的SD大鼠DRG,。
2、鏡下去除神經(jīng)根和外膜,,接種于預(yù)先涂有L-多聚賴氨酸的96孔培養(yǎng)板中,,每孔1個(gè)DRG。
3,、待DRG貼壁后加入100μl無血清L15基礎(chǔ)培養(yǎng)基,,實(shí)驗(yàn)組加入純化的表達(dá)蛋白(分別以不同的蛋白濃度),陰性對照加入等體積的溶解表達(dá)蛋白的溶劑,。
4,、37℃ 5%CO2培養(yǎng),每天在Olympus倒置顯微鏡下觀察細(xì)胞的生長情況,,并進(jìn)行測量,,其數(shù)據(jù)作統(tǒng)計(jì)分析。
注意事項(xiàng)
1,、選擇適當(dāng)?shù)眉?xì)胞接種濃度,。
2、避免血清干擾:一般選小于10%的胎牛血清的培養(yǎng)液進(jìn)行試驗(yàn),。在呈色后盡量吸盡孔內(nèi)殘余培養(yǎng)液,。
3、設(shè)空白對照:與試驗(yàn)平行不加細(xì)胞只加培養(yǎng)液的空白對照,。其他試驗(yàn)步驟保持一致,,zui后比色以空白調(diào)零。
MTT實(shí)驗(yàn)吸光度zui后要在0-0.7之間,,超出這個(gè)范圍就不是直線關(guān)系,IC50是半抑制率,,意思是抑制率50%的時(shí)候藥物的濃度。把藥品稀釋成不同的濃度,,然后計(jì)算各自的抑制率,,以藥品的濃度為橫坐標(biāo),抑制率為縱坐標(biāo)作圖,,然后得到50%抑制率時(shí)候的藥品濃度,,就是IC50。要點(diǎn):藥品2倍稀釋,,多做梯度.

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