After removal of erythrocytes by dextran-promoted sedimentation, a citrate- or heparin-anticoagulated layer of whole blood or plasma is added on top of the dense Ficoll-Hypaque layer. After centrifugation, most of the lymphocytes are located at the interface between Ficoll-Hypaque and plasma.

Operation method

Program 27.1 Isolation of Lymphocytes

Principle

After removal of erythrocytes by dextran-promoted sedimentation, a citrate- or heparin-anticoagulated layer of whole blood or plasma is added on top of the dense Ficoll-Hypaque layer. After centrifugation, most of the lymphocytes are located at the interface between Ficoll-Hypaque and plasma.

Materials and Instruments

Heparin or citrate anticoagulated blood samples D-PBSA Ficoll-Hypaque
Serum-free culture solution
Syringes with blunt tip cannulae Plastic Pasteur pipettes or pipettes Hematocrit or electronic cell counter Centrifuges

Move

1. Blood specimens are collected in citrate or heparin anticoagulated containers (heparin or citrate anticoagulated blood samples, the concentration of citrate or heparin depending on the collection container. clear centrifuge tubes or plain containers) and sent to the laboratory.

2. 9 ml of blood was diluted 1:1 with D-PBSA and added to 6 ml of Ficoll-Hypaque (adjust the specific gravity to 1.077 g/cc). Use a thick clear centrifuge tube with a cap such as a 25 ml Sterilin or Nimc plain container, or add double the amount of fluid in a 50 ml clear plastic Corning centrifuge tube.

3. centrifuge (400 g, 15 min), which is the centrifugation rate measured at the center of the interface.

4. Do not agitate the interface and carefully aspirate the plasma and D-PBSA layers.

5. A syringe with a blunt tip cannula or a plastic Pasteur pipette is used to collect the liquid layer containing the lymphocytes, which are then diluted to 20 ml with a serum-free culture medium such as RPMI 1640.

6. Centrifuge the diluted cell suspension (70 g, 10 min).

7. Discard the supernatant and suspend the settled cells with 2 ml of serum-free culture medium. If several washes are required (e.g. to remove serum factor), suspend the cells in another 20 ml of serum-free culture medium and centrifuge 2 or 3 times. Finally, sediment the cells in 2 ml of serum-free culture medium.

8. Take a sample of cells, stain with methylene blue and count the nucleated cells with a blood cell counter. Take another cell sample, dissolve it in Zapoglobin (Beckman Coulter) and count the nuclei on an electronic cell counter using a 70-100 μm caliber tube.

Lymphocytes are concentrated in one layer along with some platelets and monocytes. Although most of the granulocytes centrifuged in step 3 are found primarily in the Ficoll-Hypaque, some granulocytes may be located in the lymphocyte layer. Monocytes and residual granulocytes can be removed from the lymphocyte layer by utilizing monocytes and granulocytes attached to glass (microbeads or the inner surface of the culture flask) or nylon mesh. If purer cells need to be isolated, surface markers specific to lymphocyte subpopulations can be utilized for positive sorting by MACS or FACS.

Caveat

1. Human blood can be infected with HIV, hepatitis viruses or other pathogens and should be handled with extreme caution.

2. Do not use glass Pasteur pipettes and syringes with sharp needles to add injected blood, but use 1 ml pipettes or syringes with blunt tips.

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