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.
实验注意事项:
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.