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Ficoll-Paque單個核細(xì)胞分離方法
閱讀：12134發(fā)布時間：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。
- 稀釋過的血液樣本小心鋪到Ficoll-Paque分離液上面。
注意：鋪樣本時小心不要和Ficoll-Paque分離液混合。
- 離心400g，30-40min。18°C - 20°C。
- 用無菌吸管吸掉上層血漿和血小板，不要碰到單個核細(xì)胞層（圖4，圖5）。
- 無菌吸管轉(zhuǎn)移單個核細(xì)胞層到無菌離心管。
洗滌分離細(xì)胞
1. 預(yù)估轉(zhuǎn)移的單個核細(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：高速離心會加速單個核細(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明書。
GE淋巴細(xì)胞分離液是一種無菌、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)胞等單個核細(xì)胞進(jìn)行分離。適用人外周血、骨髓和臍帶血的單個核細(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)證和美國藥典，可用于生產(chǎn)臨床級細(xì)胞治療相關(guān)產(chǎn)品。
訂購信息
品牌 | 貨號 | 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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