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Ficoll-Paque單個(gè)核細(xì)胞分離方法
閱讀：12189發(fā)布時(shí)間：2018-1-2
Ficoll-Paque產(chǎn)品是高分子量蔗糖聚合物和泛影酸鈉溶液,。Ficoll-Paque密度梯度介質(zhì)利用簡(jiǎn)單快速離心程序從少量或大量血液中有效提取高產(chǎn)和高純度的活單個(gè)核細(xì)胞,。
操作步驟：
使用前把Ficoll-Paque density gradient media加熱到18°C - 20°C。若樣本量超過(guò)3mL,，換用直徑更大的離心管,，以保證Ficoll-Paque 分離液：2.4 cm，血液樣本：3.0 cm的高度,。
樣本制備
使用新鮮血液確保所分離的單個(gè)核細(xì)胞的活性,。樣本也要加熱到18°C - 20°C。
1. 10mL離心管加入2mL抗凝血和等量的平衡鹽溶液,。
2. 顛倒或吸管混勻血液和緩沖液,。
單個(gè)核細(xì)胞分離步驟
1. Ficoll-Paque試劑瓶顛倒多次混勻。
注射器法吸取Ficoll-Paque分離液：
見圖,，去掉聚丙烯蓋,，插入注射器針頭。
吸管法吸取Ficoll-Paque分離液：
去掉聚丙烯蓋,，拉起鋁環(huán),，拉開金屬密封,，移除銀環(huán)。拿掉橡膠密封,，無(wú)菌操作吸取需要的Ficoll-Paque分離液,。
- 離心管加入3mLFicoll-Paque分離液。
- 稀釋過(guò)的血液樣本小心鋪到Ficoll-Paque分離液上面,。
注意：鋪樣本時(shí)小心不要和Ficoll-Paque分離液混合,。
- 離心400g，30-40min,。18°C - 20°C。
- 用無(wú)菌吸管吸掉上層血漿和血小板,，不要碰到單個(gè)核細(xì)胞層（圖4,，圖5）。
- 無(wú)菌吸管轉(zhuǎn)移單個(gè)核細(xì)胞層到無(wú)菌離心管,。
洗滌分離細(xì)胞
1. 預(yù)估轉(zhuǎn)移的單個(gè)核細(xì)胞的體積,。加入至少3倍體積的平衡鹽溶液（約6mL）。
2. 用吸管輕柔重懸細(xì)胞,。
3. 離心,，400-500g，10-15min,，18°C - 20°C,。
注意：高速離心會(huì)加速單個(gè)核細(xì)胞的回復(fù)。但是,，低速離心（60-100g）能去除血小板,。
4. 去掉上清。
5. 6-8mL平衡鹽溶液重懸單個(gè)核細(xì)胞,。
6. 離心,，400-500g（或60-100g），10min,，18°C - 20°C,。
7. 去上清。
8. 使用下游所需液體重懸細(xì)胞沉淀,。
附：平衡鹽溶液配制
English version of Ficoll-Paque
Ficoll-Paque product
Warm the Ficoll-Paque density gradient media to 18°C to 20°C before use. For samples larger than 3 ml, see Notes on page 8.
Preparation of the sample
Fresh blood should be used to ensure high viability of isolated mononuclear cells. Prepare the sample at 18ºC to 20°C.
1. To a 10 ml centrifuge tube add 2 ml of defibrinated- or anticoagulant-treated blood and an equal volume of balanced salt solution (final volume 4 ml).
2. Mix the blood and buffer by inverting the tube several times or by drawing the mixture in and out of a pipette.
Procedure for isolation of mononuclear cells
1. Invert the Ficoll-Paque media bottle several times to ensure thorough mixing.
For withdrawal of Ficoll-Paque media by syringe:
Snap-off the polypropylene cap and insert the syringe needle through the septum (Fig 1).
For withdrawal of Ficoll-Paque media by pipette:
Remove the snap-off polypropylene cap. Lift the aluminum ring. Pull off the metal seal. Remove the silver ring.Remove the rubber closure. Using aseptic techniques, withdraw the required volume of Ficoll-Paque media.
2. Add Ficoll-Paque media (3 ml) to the centrifuge tube.
3. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque media solution (Fig 3).
Important: When layering the sample do not mix the Ficoll-Paque media solution and the diluted blood sample.
4. Centrifuge at 400 g for 30 to 40 min at 18ºC to 20°C (brake should be turned off).
5. Draw off the upper layer containing plasma and plaets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface (Fig 4 and Fig 5). The upper layer, which contains the plasma, may be saved for later use.
6. Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette.
Washing the cell isolate
1. Estimate the volume of the transferred mononuclear cells. Add at least 3 volumes (~ 6 ml) of balanced salt solution to the mononuclear cells in the centrifuge tube.
2. Suspend the cells by gently drawing them in and out of a pipette.
3. Centrifuge at 400 to 500 × g for 10 to 15 min at 18°C to 20°C.
Note: A centrifugation at high speed increases the mononuclear cell recovery. However, if it is important to also get rid of plaets a lower centrifugation speed is recommended (60 to 100 × g).
4. Remove the supernatant.
5. Resuspend the mononuclear cells in 6 to 8 ml balanced salt solution.
6. Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of plaets) for 10 min at 18°C to 20°C.
7. Remove the supernatant.
8. Resuspend the cell pellet in media appropriate for the application.
Ficoll-Paque單個(gè)核細(xì)胞分離方法由紅榮微再翻譯整理,。準(zhǔn)確詳情以及疑難解答等參見說(shuō)明書。
GE淋巴細(xì)胞分離液是一種無(wú)菌,、即用型的淋巴細(xì)胞分離液,，根據(jù)外周血中各類細(xì)胞在密度梯度離心中呈現(xiàn)不同的密度梯度分布而將全血中的淋巴細(xì)胞等單個(gè)核細(xì)胞進(jìn)行分離。適用人外周血,、骨髓和臍帶血的單個(gè)核細(xì)胞分離,。與Ficoll-Paque PLUS不同的是,，F(xiàn)icoll-Paque PREMIUM的生產(chǎn)是在嚴(yán)格控制環(huán)境下完成的, 生產(chǎn)條件符合ISO 13485標(biāo)準(zhǔn)，GMP認(rèn)證和美國(guó)藥典,，可用于生產(chǎn)臨床級(jí)細(xì)胞治療相關(guān)產(chǎn)品,。
訂購(gòu)信息
品牌 | 貨號(hào) | 產(chǎn)品描述 | 包裝 |
GE | 17-1440-02 | Ficoll-Paque PLUS,1.078 g/ml淋巴細(xì)胞分離液 | 6 × 100 ml |
GE | 17-1440-03 | Ficoll-Paque PLUS,1.078 g/ml淋巴細(xì)胞分離液 | 6 × 500 ml |
GE | 17-5442-02 | Ficoll-Paque PREMIUM,1.077 g/ml淋巴細(xì)胞分離液 | 6 × 100 ml |
GE | 17-5442-03 | Ficoll-Paque PREMIUM,1.077 g/ml淋巴細(xì)胞分離液 | 6 × 500 ml |
GE | 17-5446-52 | Ficoll-Paque PREMIUM 1.073,1.073 g/ml 淋巴細(xì)胞分離液 | 6 × 100 ml |
咨詢GE Ficoll-Paque density gradient media 淋巴細(xì)胞分離液歡迎您致電 華雅再生醫(yī)學(xué)旗艦公司：紅榮微再（上海）生物工程技術(shù)有限公司
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