技(jì)術文獻
技術文獻
大鼠直腸組(zǔ)織(zhī)提(tí)取(qǔ)物操作流(liú)程(chéng)
1.1主要(yào)試(shì)劑(jì)與儀(yí)器:倒(dǎo)置相(xiāng)差顯微鏡(jìng)(日本(běn) olympus imt-413),co2孵箱(日(rì)本yamato it-61)。
1.2 hek-293細胞的復蘇(sū)培(péi)養(yǎng)傳代及凍(dòng)存:
1.2.1 細(xì)胞的復蘇培(péi)養(yǎng) 從(cóng)液氮罐(guàn)中取出凍存管,立即投(tóu)入(rù)37℃水(shuǐ)浴(yù),並(bìng)不(bù)斷的搖動(dòng),使之(zhī)迅(xùn)速融化。 將溶解(jiě)的(dí)細(xì)胞(bāo)凍存(cún)管取出,用酒(jiǔ)精消(xiāo)毒(dú)後打開,吸(xī)出溶(róng)有(yǒu)細(xì)胞的凍存液,加入事先裝好(hǎo)培(péi)養(yǎng)液(37℃預熱(rè),8~10倍(bèi)體積)離心管(guǎn)內,稍(shāo)微吹打混匀,然後1000rpm 5min,去除上清(qīng),加入適量(liáng)培養液,吹打(dǎ)成(chéng)細(xì)胞(bāo)懸(xuán)液, 以1×105/ml密(mì)度(dù)接(jiē)種於(yú)25cm2培養(yǎng)瓶內,在(zài)37℃,5%co2及(jí)飽(bǎo)和(hé)濕度(dù)下(xià)培(péi)養。
1.2.2 細胞傳代(dài) 原代培養(yǎng)的(dí)hek-293細胞48h換液1次,細胞長(cháng)至60%~70%融(róng)合時(shí),用(yòng)0.25%胰(yí)酶消化1~2min,將培(péi)養(yǎng)瓶再(zài)用手掌輕(qīng)輕(qīng)敲打使細(xì)胞脫壁,懸(xuán)浮,分(fēn)散,為(wéi)使細(xì)胞進一(yī)步分(fēn)散可用吹打管輕輕吹打幾(jī)次,獲得細胞懸(xuán)液,然(rán)後加入倍量(liáng)的培養(yǎng)基,溫和混匀,吸(xī)管分裝混合液(yè),傳代(dài)擴增細胞(bāo)。
1.3 細(xì)胞形(xíng)態(tài)的觀(guān)察(chá) 在(zài)倒置顯(xiǎn)微鏡下(xià)觀察(chá)並(bìng)記錄(lù)細(xì)胞(bāo)的生(shēng)長(cháng)情(qíng)況(kuàng)及形態特征。
1.4 細(xì)胞的(dí)計數 常(cháng)規消(xiāo)化細胞(bāo),待細胞(bāo)變圓,脫壁(bì)並浮(fú)起(qǐ),加(jiā)入少(shǎo)量(liáng)培養基(jī)離心(xīn),再(zài)用pbs重懸並(bìng)吹打製成細(xì)胞懸液(yè)。在細胞(bāo)計(jì)數(shù)板(bǎn)蓋玻片(piàn)的(dí)一側(cè)加微量(liáng)細(xì)胞懸液,用(yòng)10×物鏡(jìng)觀(guān)察計(jì)數板(bǎn)四(sì)角(jiǎo)大方(fāng)格細胞(bāo)數(shù),按公式(shì)計(jì)算出細(xì)胞數(shù)。細(xì)胞(bāo)數/ml原(yuán)液=(四方(fāng)格(gé)細(xì)胞(bāo)數(shù)之和(hé)/4) ×104。
1.5 細胞的貼(tiē)壁率(shuài) 指數生長期(qī)的細(xì)胞(bāo),以(yǐ)0.25%胰(yí)酶(méi)消(xiāo)化(huà)成單細胞(bāo)懸液,計(jì)數後分(fēn)別接種於12個25cm2培養瓶(píng)中(zhōng),每兩小時隨(suí)機取(qǔ)出(chū)3瓶(píng)細(xì)胞,吸除(chú)培(péi)養基(jī)及(jí)未(wèi)貼壁細胞,加入胰消(xiāo)化(huà)酶消化,計數已(yǐ)貼壁細胞(bāo),取其(qí)平(píng)均值(zhí),按照(zhào)公(gōng)式:貼(tiē)壁(bì)率(%)=(已貼(tiē)壁細胞(bāo)數(shù)/接(jiē)種細胞數) ×100%,計算貼壁率(shuài)。
1.6 生長曲(qū)綫 取指(zhǐ)數生長期的細(xì)胞(bāo)用胰酶消(xiāo)化(huà)製(zhì)成(chéng)單細胞懸(xuán)液,以(yǐ)1×104/孔接種於24孔板,每(měi)孔(kǒng)加(jiā)入(rù)1ml培(péi)養基。每(měi)天隨(suí)機取3孔(kǒng),計數(shù)每孔(kǒng)細(xì)胞的數目並(bìng)計算平(píng)均值(zhí)。連(lián)續計數10d,繪製細(xì)胞(bāo)生長曲(qū)綫。
羊抗人IgA (抗(kàng)血(xiě)清) Anti-Human IgA antibody produced in goat 1ML 免(miǎn)疫血清(qīng)
兔(tù)抗人IgA (抗(kàng)血(xiě)清) Anti-Human IgA antibody produced in ra... 1ML 免疫血清
兔(tù)抗(kàng)人IgG~Fc (抗血清) Anti-Human IgG (Fc spec.) produced in ... 1ML 免疫(yì)血清
羊抗人(rén)IgG (抗(kàng)血(xiě)清) Anti-Human IgG antibody produced in goat 1ML 免(miǎn)疫血清
兔抗(kàng)人IgG (抗(kàng)血清) Anti-Human IgG antibody produced in ra... 1ML 免疫(yì)血清(qīng)
羊抗(kàng)人(rén)IgM血(xiě)清 (μ鏈(liàn)特異) Anti-Human IgM (μ-chain spec.) produc... 1ML 免疫血(xiě)清
羊抗(kàng)人IgM (抗(kàng)血清(qīng)) Anti-Human IgM antibody produced in goat 1ML 免(miǎn)疫血清
兔抗人IgM (抗血(xiě)清(qīng)) Anti-Human IgM antibody produced in ra... 1ML 免疫血清(qīng)
羊抗人(rén)血(xiě)清(qīng) (多價抗(kàng)體) Anti-Human Serum antibody produced in ... 1ML 免(miǎn)疫(yì)血清(qīng)
兔(tù)抗人(rén)血(xiě)清(qīng) (多價抗體(tǐ)) Anti-Human Serum antibody produced in ... 1ML 免(miǎn)疫血清
兔抗猴(hóu)IgG (抗血清) Anti-Monkey IgG antibody produced in r... 1ML 免(miǎn)疫(yì)血清
兔抗(kàng)小(xiǎo)鼠IgG~Fc (抗血清) Anti-Mouse IgG (Fc spec.) produced in ... 1ML 免疫(yì)血清(qīng)
羊抗(kàng)小鼠IgG (抗血清) Anti-Mouse IgG antibody produced in goat 1ML 免疫血(xiě)清
兔(tù)抗小鼠IgG (抗(kàng)血清) Anti-Mouse IgG antibody produced in ra... 1ML 免疫(yì)血(xiě)清(qīng)
兔抗小(xiǎo)鼠血清 (多價抗(kàng)體) Anti-Mouse Serum antibody produced in ... 1ML 免(miǎn)疫(yì)血(xiě)清(qīng)
兔(tù)抗(kàng)HRP (抗血清(qīng)) Anti-Peroxidase antibody produced in r... 1ML 免疫血(xiě)清
羊抗(kàng)豬IgG (抗(kàng)血清(qīng)) Anti-Pig IgG antibody produced in goat 1ML 免疫血(xiě)清
羊抗兔(tù)IgG (抗(kàng)血(xiě)清(qīng)) Anti-Rabbit IgG antibody produced in g... 1ML 免(miǎn)疫血清(qīng)
羊抗(kàng)大(dà)鼠IgG (抗(kàng)血清) Anti-Rat IgG antibody produced in goat 1ML 免疫(yì)血清(qīng)
兔(tù)抗(kàng)大鼠(shǔ)IgG (抗(kàng)血清) Anti-Rat IgG antibody produced in rabbit 1ML 免(miǎn)疫血(xiě)清(qīng)
兔抗綿羊(yáng)IgG (抗血(xiě)清(qīng)) Anti-Sheep IgG antibody produced in ra... 1ML 免疫(yì)血清(qīng)
圖標(biāo) FITC熒光(guāng)標(biāo)記抗體;免疫組化(huà)/免疫(yì)熒光/流(liú)式(shì)熒光:1:20;抗體含量(liáng):10mg/ml;-20 ℃ 保(bǎo)存(cún)
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