技術(shù)文獻(xiàn)
技術(shù)文獻(xiàn)
1.1主要試劑與儀器:倒置相差顯微鏡(日本 olympus imt-413),co2孵箱(日本yamato it-61)。
1.2 hek-293細(xì)胞的復(fù)蘇培養(yǎng)傳代及凍存:
1.2.1 細(xì)胞的復(fù)蘇培養(yǎng) 從液氮罐中取出凍存管,立即投入37℃水浴,并不斷的搖動(dòng),使之迅速融化。 將溶解的細(xì)胞凍存管取出,用酒精消毒后打開,吸出溶有細(xì)胞的凍存液,加入事先裝好培養(yǎng)液(37℃預(yù)熱,8~10倍體積)離心管內(nèi),稍微吹打混勻,然后1000rpm 5min,去除上清,加入適量培養(yǎng)液,吹打成細(xì)胞懸液, 以1×105/ml密度接種于25cm2培養(yǎng)瓶?jī)?nèi),在37℃、5%co2及飽和濕度下培養(yǎng)。
1.2.2 細(xì)胞傳代 原代培養(yǎng)的hek-293細(xì)胞48h換液1次,細(xì)胞長(zhǎng)至60%~70%融合時(shí),用0.25%胰酶消化1~2min,將培養(yǎng)瓶再用手掌輕輕敲打使細(xì)胞脫壁、懸浮、分散,為使細(xì)胞進(jìn)一步分散可用吹打管輕輕吹打幾次,獲得細(xì)胞懸液,然后加入倍量的培養(yǎng)基,溫和混勻,吸管分裝混合液,傳代擴(kuò)增細(xì)胞。
1.3 細(xì)胞形態(tài)的觀察 在倒置顯微鏡下觀察并記錄細(xì)胞的生長(zhǎng)情況及形態(tài)特征。
1.4 細(xì)胞的計(jì)數(shù) 常規(guī)消化細(xì)胞,待細(xì)胞變圓、脫壁并浮起,加入少量培養(yǎng)基離心,再用pbs重懸并吹打制成細(xì)胞懸液。在細(xì)胞計(jì)數(shù)板蓋玻片的一側(cè)加微量細(xì)胞懸液,用10×物鏡觀察計(jì)數(shù)板四角大方格細(xì)胞數(shù),按公式計(jì)算出細(xì)胞數(shù)。細(xì)胞數(shù)/ml原液=(四方格細(xì)胞數(shù)之和/4) ×104。
1.5 細(xì)胞的貼壁率 指數(shù)生長(zhǎng)期的細(xì)胞,以0.25%胰酶消化成單細(xì)胞懸液,計(jì)數(shù)后分別接種于12個(gè)25cm2培養(yǎng)瓶中,每?jī)尚r(shí)隨機(jī)取出3瓶細(xì)胞,吸除培養(yǎng)基及未貼壁細(xì)胞,加入胰消化酶消化、計(jì)數(shù)已貼壁細(xì)胞,取其平均值,按照公式:貼壁率(%)=(已貼壁細(xì)胞數(shù)/接種細(xì)胞數(shù)) ×100%,計(jì)算貼壁率。
1.6 生長(zhǎng)曲線 取指數(shù)生長(zhǎng)期的細(xì)胞用胰酶消化制成單細(xì)胞懸液,以1×104/孔接種于24孔板,每孔加入1ml培養(yǎng)基。每天隨機(jī)取3孔,計(jì)數(shù)每孔細(xì)胞的數(shù)目并計(jì)算平均值。連續(xù)計(jì)數(shù)10d,繪制細(xì)胞生長(zhǎng)曲線。
羊抗人IgA (抗血清) Anti-Human IgA antibody produced in goat 1ML 免疫血清
兔抗人IgA (抗血清) Anti-Human IgA antibody produced in ra... 1ML 免疫血清
兔抗人IgG~Fc (抗血清) Anti-Human IgG (Fc spec.) produced in ... 1ML 免疫血清
羊抗人IgG (抗血清) Anti-Human IgG antibody produced in goat 1ML 免疫血清
兔抗人IgG (抗血清) Anti-Human IgG antibody produced in ra... 1ML 免疫血清
羊抗人IgM血清 (μ鏈特異) Anti-Human IgM (μ-chain spec.) produc... 1ML 免疫血清
羊抗人IgM (抗血清) Anti-Human IgM antibody produced in goat 1ML 免疫血清
兔抗人IgM (抗血清) Anti-Human IgM antibody produced in ra... 1ML 免疫血清
羊抗人血清 (多價(jià)抗體) Anti-Human Serum antibody produced in ... 1ML 免疫血清
兔抗人血清 (多價(jià)抗體) Anti-Human Serum antibody produced in ... 1ML 免疫血清
兔抗猴IgG (抗血清) Anti-Monkey IgG antibody produced in r... 1ML 免疫血清
兔抗小鼠IgG~Fc (抗血清) Anti-Mouse IgG (Fc spec.) produced in ... 1ML 免疫血清
羊抗小鼠IgG (抗血清) Anti-Mouse IgG antibody produced in goat 1ML 免疫血清
兔抗小鼠IgG (抗血清) Anti-Mouse IgG antibody produced in ra... 1ML 免疫血清
兔抗小鼠血清 (多價(jià)抗體) Anti-Mouse Serum antibody produced in ... 1ML 免疫血清
兔抗HRP (抗血清) Anti-Peroxidase antibody produced in r... 1ML 免疫血清
羊抗豬IgG (抗血清) Anti-Pig IgG antibody produced in goat 1ML 免疫血清
羊抗兔IgG (抗血清) Anti-Rabbit IgG antibody produced in g... 1ML 免疫血清
羊抗大鼠IgG (抗血清) Anti-Rat IgG antibody produced in goat 1ML 免疫血清
兔抗大鼠IgG (抗血清) Anti-Rat IgG antibody produced in rabbit 1ML 免疫血清
兔抗綿羊IgG (抗血清) Anti-Sheep IgG antibody produced in ra... 1ML 免疫血清
圖標(biāo) FITC熒光標(biāo)記抗體;免疫組化/免疫熒光/流式熒光:1:20;抗體含量:10mg/ml;-20 ℃ 保存
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