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兔外周血單核細胞分離液試劑盒
貨號：P5320
規格：2×200ml/KIT
保(bao)存(cun) ：室溫避光儲存(cun)，有效期(qi)少 2 年。

兔外周血單核細胞分離液試劑盒

操作(zuo)(zuo)步驟： 1. 取新鮮抗(kang)(kang)凝全(quan)血(xue)（EDTA、枸櫞酸(suan)鈉或(huo)肝素抗(kang)(kang)凝血(xue)均(jun)可(ke)(ke)）或(huo)者(zhe)去纖(xian)維(wei)蛋白血(xue)液(ye)(ye)(ye)(ye)(ye)，用(yong)(yong)(yong)(yong)等體(ti)積(ji)(ji)的(de)(de)(de)(de)全(quan)血(xue)及組織稀(xi)(xi)(xi)釋(shi)液(ye)(ye)(ye)(ye)(ye)或(huo)者(zhe)PBS稀(xi)(xi)(xi)釋(shi)全(quan)血(xue)。 2. 取一支(zhi)無菌離(li)心(xin)(xin)管，先加(jia)入試(shi)(shi)(shi)劑A，再加(jia)入試(shi)(shi)(shi)劑D，使(shi)兩者(zhe)形成梯(ti)度(du)(du)界面(mian)（試(shi)(shi)(shi)劑A：試(shi)(shi)(shi)劑D的(de)(de)(de)(de)體(ti)積(ji)(ji)比為(wei)3：2，如(ru)稀(xi)(xi)(xi)釋(shi)后(hou)的(de)(de)(de)(de)血(xue)液(ye)(ye)(ye)(ye)(ye)樣(yang)本(ben)小(xiao)于5ml，則先后(hou)加(jia)入3ml試(shi)(shi)(shi)劑A和(he)(he)2ml試(shi)(shi)(shi)劑D；如(ru)稀(xi)(xi)(xi)釋(shi)后(hou)的(de)(de)(de)(de)血(xue)液(ye)(ye)(ye)(ye)(ye)樣(yang)本(ben)大(da)于5ml，試(shi)(shi)(shi)劑總(zong)量與稀(xi)(xi)(xi)釋(shi)后(hou)的(de)(de)(de)(de)血(xue)液(ye)(ye)(ye)(ye)(ye)樣(yang)本(ben)量相等），兩種(zhong)試(shi)(shi)(shi)劑的(de)(de)(de)(de)分(fen)層(ceng)一定(ding)要(yao)清晰(xi)。 3. 將(jiang)稀(xi)(xi)(xi)釋(shi)后(hou)的(de)(de)(de)(de)血(xue)液(ye)(ye)(ye)(ye)(ye)平(ping)鋪到分(fen)離(li)液(ye)(ye)(ye)(ye)(ye)液(ye)(ye)(ye)(ye)(ye)面(mian)上方，注意(yi)保持兩液(ye)(ye)(ye)(ye)(ye)面(mian)界面(mian)清晰(xi)。（可(ke)(ke)以(yi)(yi)(yi)使(shi)用(yong)(yong)(yong)(yong)巴(ba)氏德吸管吸取血(xue)液(ye)(ye)(ye)(ye)(ye)，然后(hou)將(jiang)血(xue)液(ye)(ye)(ye)(ye)(ye)小(xiao)心(xin)(xin)的(de)(de)(de)(de)平(ping)鋪于分(fen)離(li)液(ye)(ye)(ye)(ye)(ye)上，因(yin)為(wei)兩者(zhe)的(de)(de)(de)(de)密(mi)度(du)(du)差異，將(jiang)形成明(ming)(ming)顯(xian)的(de)(de)(de)(de)分(fen)層(ceng)界面(mian)。如(ru)果樣(yang)品較(jiao)多，加(jia)樣(yang)的(de)(de)(de)(de)時間(jian)較(jiao)長(chang)，在離(li)心(xin)(xin)之前(qian)出現(xian)紅(hong)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)成團下沉屬正常現(xian)象） 4. 室溫，水平(ping)轉(zhuan)子500~800g，離(li)心(xin)(xin)20~30min（血(xue)液(ye)(ye)(ye)(ye)(ye)的(de)(de)(de)(de)體(ti)積(ji)(ji)越(yue)(yue)大(da)所需的(de)(de)(de)(de)離(li)心(xin)(xin)力越(yue)(yue)大(da)，離(li)心(xin)(xin)時間(jian)越(yue)(yue)長(chang)，分(fen)離(li)條件(jian)需自行摸索(suo)，離(li)心(xin)(xin)轉(zhuan)速不(bu)(bu)超過1000g）。 5. 離(li)心(xin)(xin)后(hou)將(jiang)出現(xian)明(ming)(ming)顯(xian)的(de)(de)(de)(de)分(fen)層(ceng)： 層(ceng)為(wei)血(xue)漿層(ceng)；第(di)(di)(di)二層(ceng)為(wei)白色單(dan)核細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)層(ceng)；第(di)(di)(di)三(san)層(ceng)為(wei)透明(ming)(ming)試(shi)(shi)(shi)劑D液(ye)(ye)(ye)(ye)(ye)層(ceng)；第(di)(di)(di)四(si)層(ceng)為(wei)半透明(ming)(ming)試(shi)(shi)(shi)劑A液(ye)(ye)(ye)(ye)(ye)層(ceng)；第(di)(di)(di)五層(ceng)為(wei)紅(hong)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)層(ceng)（如(ru)圖示）。 6. 小(xiao)心(xin)(xin)吸取第(di)(di)(di)二層(ceng)試(shi)(shi)(shi)劑D層(ceng)和(he)(he)第(di)(di)(di)三(san)層(ceng)乳白色細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)層(ceng)到另一無菌的(de)(de)(de)(de)15ml離(li)心(xin)(xin)管中，加(jia)入10mL細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)洗滌液(ye)(ye)(ye)(ye)(ye)或(huo)PBS，顛倒(dao)混勻(yun)，250g，離(li)心(xin)(xin)10min。 7. 棄(qi)上清，5mL的(de)(de)(de)(de)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)清洗液(ye)(ye)(ye)(ye)(ye)或(huo)PBS重懸(xuan)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)，250g，離(li)心(xin)(xin)10min。 8. 重復(fu)步驟7 9. 棄(qi)上清，細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)重懸(xuan)備用(yong)(yong)(yong)(yong)。 10. 差異貼壁法(fa)純(chun)化細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)：用(yong)(yong)(yong)(yong)單(dan)核細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)培養基以(yi)(yi)(yi)10 6個/ml的(de)(de)(de)(de)密(mi)度(du)(du)重懸(xuan)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)，將(jiang)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)鋪于細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)板或(huo)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)瓶中，置于細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)培養箱中進行貼壁培養。 1) 2~4 h內貼壁的(de)(de)(de)(de)為(wei)單(dan)核細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)。 2) 10~24 h內貼壁的(de)(de)(de)(de)單(dan)個核細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)為(wei)內皮、內皮祖細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)、干細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao) 。 3) 不(bu)(bu)貼壁的(de)(de)(de)(de)為(wei)淋巴(ba)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)。根據不(bu)(bu)同(tong)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)的(de)(de)(de)(de)貼壁時間(jian)存在差異，以(yi)(yi)(yi)達到簡(jian)單(dan)純(chun)化單(dan)核細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)的(de)(de)(de)(de)目的(de)(de)(de)(de)。此法(fa)成本(ben)低，操作(zuo)(zuo)簡(jian)便(bian)。如(ru)需獲(huo)得高純(chun)度(du)(du)的(de)(de)(de)(de)目的(de)(de)(de)(de)細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)則需選用(yong)(yong)(yong)(yong)免疫磁珠分(fen)選或(huo)者(zhe)是應用(yong)(yong)(yong)(yong)流式技術分(fen)選細(xi)(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)