Ficoll-Paque产品是高分子量蔗糖聚合物和泛影酸钠溶液。Ficoll-Paque密度梯度介质利用简单快速离心程序从少量或大量血液中有效提取高产和高纯度的活单个核细胞。

操作步骤：

使用前把Ficoll-Paque density gradient media加热到18°C - 20°C。若样本量超过3mL，换用直径更大的离心管，以保证Ficoll-Paque 分离液：2.4 cm，血液样本：3.0 cm的高度。

样本制备

使用新鲜血液确保所分离的单个核细胞的活性。样本也要加热到18°C - 20°C。

1. 10mL离心管加入2mL抗凝血和等量的平衡盐溶液。

2. 颠倒或吸管混匀血液和缓冲液。

单个核细胞分离步骤

1. Ficoll-Paque试剂瓶颠倒多次混匀。

注射器法吸取Ficoll-Paque分离液：

见图，去掉聚丙烯盖，插入注射器针头。

吸管法吸取Ficoll-Paque分离液：

去掉聚丙烯盖，拉起铝环，拉开金属密封，移除银环。拿掉橡胶密封，无菌操作吸取需要的Ficoll-Paque分离液。

• 离心管加入3mLFicoll-Paque分离液。
• 稀释过的血液样本小心铺到Ficoll-Paque分离液上面。

注意：铺样本时小心不要和Ficoll-Paque分离液混合。

• 离心400g，30-40min。18°C - 20°C。
• 用无菌吸管吸掉上层血浆和血小板，不要碰到单个核细胞层（图4，图5）。
• 无菌吸管转移单个核细胞层到无菌离心管。

洗涤分离细胞

1. 预估转移的单个核细胞的体积。加入至少3倍体积的平衡盐溶液（约6mL）。

2. 用吸管轻柔重悬细胞。

3. 离心，400-500g，10-15min，18°C - 20°C。

注意：高速离心会加速单个核细胞的回复。但是，低速离心（60-100g）能去除血小板。

4. 去掉上清。

5. 6-8mL平衡盐溶液重悬单个核细胞。

6. 离心，400-500g（或60-100g），10min，18°C - 20°C。

7. 去上清。

8. 使用下游所需液体重悬细胞沉淀。

附：平衡盐溶液配制

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单个核细胞分离方法由红荣微再翻译整理。准确详情以及疑难解答等参见说明书。

GE淋巴细胞分离液是一种无菌、即用型的淋巴细胞分离液，根据外周血中各类细胞在密度梯度离心中呈现不同的密度梯度分布而将全血中的淋巴细胞等单个核细胞进行分离。适用人外周血、骨髓和脐带血的单个核细胞分离。与Ficoll-Paque PLUS不同的是，Ficoll-Paque PREMIUM的生产是在严格控制环境下完成的, 生产条件符合ISO 13485标准，GMP认证和美国药典，可用于生产临床级细胞治疗相关产品。

订购信息

咨询GE Ficoll-Paque density gradient media 淋巴细胞分离液欢迎您致电 华雅再生医学旗舰公司：红荣微再（上海）生物工程技术有限公司