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Ficoll-Paque單個核細胞分離方法

更新時間:2018-01-02瀏覽:11603次

 

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(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,不要碰到單個核細胞層(圖4,圖5)。
  • 無菌吸管轉(zhuǎn)移單個核細胞層到無菌離心管。

 

洗滌分離細胞

1. 預估轉(zhuǎn)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(jù)外周血中各類細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,F(xiàn)icoll-Paque PREMIUM的生產(chǎn)是在嚴格控制環(huán)境下完成的, 生產(chǎn)條件符合ISO 13485標準,GMP認證和美國藥典,可用于生產(chǎn)臨床級細胞治療相關產(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淋巴細胞分離液

6 × 100 ml

GE

17-1440-03

Ficoll-Paque PLUS,1.078 g/ml淋巴細胞分離液

6 × 500 ml

GE

17-5442-02

Ficoll-Paque PREMIUM,1.077 g/ml淋巴細胞分離液

6 × 100 ml

GE

17-5442-03

Ficoll-Paque PREMIUM,1.077 g/ml淋巴細胞分離液

6 × 500 ml

GE

17-5446-52

Ficoll-Paque PREMIUM 1.073,1.073 g/ml 淋巴細胞分離液

6 × 100 ml

 

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(yī)學旗艦公司:紅榮微再(上海)生物工程技術有限公司