细胞密度梯度分离介质Ficoll：人外周血单个核细胞的分离
Cytiva（思拓凡）
10019
Ficoll：一种已灭菌的，特定密度的密度梯度离心介质。被广泛用作从外周血、骨髓及脐带血中分离高活性的单个核细胞。
Ficoll-Paque溶液包含：
Ficoll 产品细分如下：
使用Ficoll (1.077g/ml) 分离人外周血单个核细胞:
原理：人外周血单个核细胞(peripheral blood mononuclearcell, PBMC)包括淋巴细胞和单核细胞，其体积、形状和比重与其他细胞不同，红细胞和多核白细胞比重较大，为1.092左右，而淋巴细胞和单个核细胞比重为1.075左右。利用密度在1.077±0.001g/L之间近于等渗的淋巴细胞分离液作密度梯度离心时，各种血液成分将按密度梯度重新分布聚集。血浆和血小板由于密度较低, 故悬浮于分层液的上部；红细胞与粒细胞由于密度较大, 故沉于分层液的底部；PBMC密度稍低于分层液，故位于分层液界面上，这样就可获得PBMC（如下图所示）。
实验操作步骤：
样品制备：
单个核细胞细胞回收：
Ficoll-Paque溶液包含：
- Ficoll PM400：由高分子量蔗糖（Mr 400K）聚合物与表氯醇聚合合成(凝集红细胞)
- 泛影酸钠：使溶液在低粘度下具有高密度
- EDTA二钠钙
Ficoll 产品细分如下：
使用Ficoll (1.077g/ml) 分离人外周血单个核细胞:
原理：人外周血单个核细胞(peripheral blood mononuclearcell, PBMC)包括淋巴细胞和单核细胞，其体积、形状和比重与其他细胞不同，红细胞和多核白细胞比重较大，为1.092左右，而淋巴细胞和单个核细胞比重为1.075左右。利用密度在1.077±0.001g/L之间近于等渗的淋巴细胞分离液作密度梯度离心时，各种血液成分将按密度梯度重新分布聚集。血浆和血小板由于密度较低, 故悬浮于分层液的上部；红细胞与粒细胞由于密度较大, 故沉于分层液的底部；PBMC密度稍低于分层液，故位于分层液界面上，这样就可获得PBMC（如下图所示）。
实验操作步骤：
样品制备：
1. To a 10 ml test-tube add 2 ml of defibrinated or anticoagulant-treated blood and an equal volume of balanced salt solution. (Final volume 4 ml.)
2. Mix by drawing the blood and buffer in and out of a Pasteur pipette.
单个核细胞的分离：
1. Invert the Ficoll-Paque PLUS bottle several times to ensure thorough mixing.
2. Add Ficoll-Paque PLUS (3 ml) to the centrifuge tube.
Important: When layering the sample do not mix Ficoll- Paque PLUS and the diluted blood sample.
3. Carefully layer the diluted blood sample (4 ml) on Ficoll-Paque PLUS
4. Centrifuge at 400 × g for 30-40 minutes at 18–20 °C.
5. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface (Fig 1, 2). Care should be taken not to disturb the lymphocyte layer. The upper layer of plasma, which is essentially free of cells, may be saved for later use.
单个核细胞细胞回收：
- Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube.
- Add at least 3 volumes (6 ml) of balanced salt solution to the lymphocytes in the test tube.
- Suspend the cells by gently drawing them in and out of a Pasteur pipette.
- Centrifuge at 60–100 × g for 10 minutes at 18–20 °C.
- Remove the supernatant.
- Suspend the lymphocytes in 6–8 ml balanced salt solution by gently drawing them in and out of the Pasteur pipette.
- Centrifuge at 60–100 × g for 10 minutes at 18–20 °C.
- Remove the supernatant.
- The lymphocytes should now be suspended in the medium appropriate to the application.
- 温度（18–20 °C）很关键！
- 水平转子离心机（制动关闭）
- 温度过低：如果被冷冻，请溶化后颠倒混匀
- 温度过高：Aggregation of erythrocytes is enhanced at higher temperatures (37°C), which consequently decreases the yield of mononuclear cells. And It is known that the densities of Ficoll-Paque products decrease with increase in temperature. For instance Ficoll-Paque PREMIUM (1.077 g/ml) exhibits a density of 1.0772, 1.0767, and 1.0758 at 20ºC,22ºC, and 25ºC, respectively.
- 血液样品需要新鲜（24hr内请使用）
