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MTT檢測(cè)|實(shí)驗(yàn)技術(shù)服務(wù)

時(shí)間:2015/6/2閱讀:396
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關(guān)鍵詞:MTT檢測(cè)|實(shí)驗(yàn)技術(shù)服務(wù)
簡(jiǎn)介:世界*品牌MTT檢測(cè)|實(shí)驗(yàn)技術(shù)服務(wù)原裝,,質(zhì)量保證,*。
MTT檢測(cè)|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(huì)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(cè)序?qū)嶒?yàn)流程:
MTT分析法以活細(xì)胞代謝物還原劑3-(4,5)-dimethylthiahiazo (-z-y1)-3,,5-di- phenytetrazoliumromide,, MTT噻唑藍(lán)為基礎(chǔ)。MTT為黃色化合物,,是一種接受氫離子的染料,,可作用于活細(xì)胞線粒體中的呼吸鏈,在琥珀酸脫氫酶和細(xì)胞色素C的作用下tetrazolium環(huán)開裂,,生成藍(lán)色的formazan結(jié)晶,,formazan結(jié)晶的生成量?jī)H與活細(xì)胞數(shù)目成正比(死細(xì)胞中琥珀酸脫氫酶消失,不能將MTT還原),。還原生成的formazan結(jié)晶可在含50%的N,,N-二甲基甲酰胺和20%的十二甲基磺酸鈉(pH 4.7)的MTT溶解液中溶解,利用酶標(biāo)儀測(cè)定490 nm處的光密度OD值,,以反映出活細(xì)胞數(shù)目,。也可以用DMSO來溶解。
1,、接種細(xì)胞
用含10%胎小牛血清得培養(yǎng)液配成單個(gè)細(xì)胞懸液,,以每孔1000-10000個(gè)細(xì)胞接種到96孔板,每孔體積200ul,。
2,、培養(yǎng)細(xì)胞
同一般培養(yǎng)條件,培養(yǎng)3-5天(可根據(jù)試驗(yàn)?zāi)康暮鸵鬀Q定培養(yǎng)時(shí)間),。
3,、呈色
培養(yǎng)3-5天后,每孔加MTT溶液(5mg/ml用PBS 配)20ul.繼續(xù)孵育4小時(shí),,終止培養(yǎng),,小心吸棄孔內(nèi)培養(yǎng)上清液,對(duì)于懸浮細(xì)胞需要離心后再吸棄孔內(nèi)培養(yǎng)上清液,。每孔加150ul DMSO,,振蕩10分鐘,使結(jié)晶物充分融解,。
4,、比色
選擇490nm波長(zhǎng),在酶聯(lián)免疫監(jiān)測(cè)儀上測(cè)定各孔光吸收值,,記錄結(jié)果,,以時(shí)間為橫坐標(biāo),,吸光值為縱坐標(biāo)繪制細(xì)胞生長(zhǎng)曲線。
二,、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(duì)照:與試驗(yàn)平行不加細(xì)胞只加培養(yǎng)液的空白對(duì)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,,做點(diǎn)線圖即可!

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