Scheme of lymphocyte separation by ficoll

1. Prepare a fresh heparinized blood sample (e.g: 3 ml).

2. Dilute the whole blood sample with equal volume of saline (PBS: Phosphate Buffer
Saline) (e.g: 3 ml whole blood + 3 ml saline) in a falcon tube.

3. In another falcon tube, add half the volume of the diluted blood (e.g: for the 6 ml
diluted blood add 3 ml ficoll), then using Pasteur or automatic pipette start
titrating the blood over the ficoll slowly drop by drop on the falcon borders
(walls) without breaking through the ficoll layer.
Note: your falcon tube has to be in 45 degree angle, titration on the blood must
be on the falcon tip in perpendicular position.

4. After that take the falcon for centrifugation (R.T, 1800rpm for about 25-30 min.).

5. After centrifugation, you get 4 layered sample (plasma, buffy coat, ficoll and then
granulocytes).

6. Take the buffy coat out in a new falcon tube add 10 saline and centrifugate (R.T,
2000 rpm for 5-7 min).

7. Discard the formed supernatant, add 5 ml saline to the lymphocyte palette at the
bottom wash another one or two times with same washing conditions as (step
6).
Note: If you got RBCs pelleted with the lymphocytes, add 2-3 ml ammonium
chloride, mix them with the palette and incubate them for 3-5 min, centrifugate,
discard and continue washing again.
8. To the final palette, resuspend it in 1 ml saline, mix well and count the cells under
the microscope using hemocytometer.