Ficoll-Paque單個核細胞分離方法

Ficoll-Paque產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和高純度的活單個核細胞。

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，拉開金屬密封，移除銀環。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，不要碰到單個核細胞層（圖4，圖5）。
• 無菌吸管轉移單個核細胞層到無菌離心管。

洗滌分離細胞

1. 預估轉移的單個核細胞的體積。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胞的回復。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單個核細胞分離方法由紅榮微再翻譯整理。準確詳情以及疑難解答等參見說明書。

GE淋巴細胞分離液是一種無菌、即用型的淋巴細胞分離液，根據外周血中各類細胞在密度梯度離心中呈現不同的密度梯度分布而將全血中的淋巴細胞等單個核細胞進行分離。適用人外周血、骨髓和臍帶血的單個核細胞分離。與Ficoll-Paque PLUS不同的是，Ficoll-Paque PREMIUM的生產是在嚴格控制環境下完成的, 生產條件符合ISO 13485標準，GMP認證和美國藥典，可用于生產臨床級細胞治療相關產品。

訂購信息

咨詢GE Ficoll-Paque density gradient media 淋巴細胞分離液歡迎您致電 華雅再生醫學旗艦公司：紅榮微再（上海）生物工程技術有限公司