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SIGMA現(xiàn)貨,SIGMA代理

研究T細(xì)胞產(chǎn)生的細(xì)胞因子的方法

時(shí)間:2011/10/13閱讀:785
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乾思生物編輯整理:
1)研究細(xì)胞因子mRNA的方法
絕大多數(shù)細(xì)胞因子mRNA在T細(xì)胞中只短暫存在,其存在的量與T細(xì)胞產(chǎn)生細(xì)胞因子的量也有很好的相關(guān)性。所以,在大多數(shù)情況下,細(xì)胞內(nèi)mRNA的量可以反映細(xì)胞產(chǎn)生細(xì)胞因子的情況。
檢測(cè)細(xì)胞因子mRNA的試驗(yàn)主要有:競(jìng)爭(zhēng)性聚合酶鏈反應(yīng)、Northern印跡雜交、斑點(diǎn)雜交、逆轉(zhuǎn)錄聚合酶鏈反應(yīng)、原位雜交。
2)研究細(xì)胞因子的免疫檢測(cè)法
免疫檢測(cè)法主要有放射免疫檢測(cè)法、酶免疫測(cè)定法、免疫熒光測(cè)定法和免疫發(fā)光測(cè)定法等。
3)研究細(xì)胞因子的生物學(xué)方法
1.用含10%小牛血清的RPMI-1640培養(yǎng)液培養(yǎng)HT2細(xì)胞。取對(duì)數(shù)生長(zhǎng)期細(xì)胞,用細(xì)胞培養(yǎng)液洗滌細(xì)胞2次,每次離心1000×g 10分鐘。
2.取96孔細(xì)胞培養(yǎng)液,每孔加0.1ml含4×103 HT2細(xì)胞的細(xì)胞培養(yǎng)液。再加0.1ml T細(xì)胞培養(yǎng)上清液或經(jīng)系列稀釋的IL-2標(biāo)準(zhǔn)品,每個(gè)稀釋度重復(fù)3孔。在37℃ 5% CO2的飽和濕度二氧化碳培養(yǎng)箱中培養(yǎng)24小時(shí)。
3.每孔加10μCi(3.7×105Bq)[3H]脫氧胸苷(10μl),在37℃ 5% CO2的飽和濕度二氧化碳培養(yǎng)箱中繼續(xù)培養(yǎng)4~8小時(shí)。
4.用多孔收集器將細(xì)胞收集到玻璃纖維濾紙上,在液體閃爍計(jì)數(shù)儀中計(jì)數(shù)每分鐘放射活性(cpm)。用cpm對(duì)稀釋度作圖,根據(jù)標(biāo)準(zhǔn)曲線判定T細(xì)胞培養(yǎng)上清液中IL-2的含量。

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