Peripheral Blood Lymphocyte Preparation

1. Transfer 30 mL of the peripheral blood to two 50-mL conical tubes and add 20 mL of PBS solution to each tube. Cap the tubes and mix the contents gently by inverting tubes several times.

2. Place the tubes in a centrifuge and spin at 300g for 15 min at room temperature.

3. Carefully aspirate and discard two thirds of the top plasma layer (supernatant) without disturbing the underlying cell mass.

4. Replace the discarded plasma with an equal volume of PBS buffer solution, cap the tubes and mix well by gently inverting the tubes.

5. Mix well the contents of the Ficoll bottle, wipe the rubber cap with 70% ethanol then inject approx 15 mL air with a 20-mL syringe and needle and withdraw 20 mL of the Ficoll solution. Equally divide the Ficoll solution between two new 50-mL Falcon tubes.

6. Layer the blood mixture on top of the Ficoll solution using a 10-mL pipet and pipettor. Proceed carefully so as to not disturb the Ficoll-blood interface by keeping the pipet tip against the tube wall close to the blood surface. The Ficoll to blood volume should be approx 2:3.

7. Place the tubes with the layered blood on Ficoll in the centrifuge. Centrifuge for 30 min at 350g, room temperature (with the brake off).

8. Using a 10-mL pipet and pipetor, carefully aspirate the plasma layer from the Ficoll tubes and discard. Leave approx 5 mL of plasma on top of the mononuclear cell layer to avoid cell loss. Carefully aspirate the mononuclear cell layer and transfer to two new 50-mL Falcon tubes. A total of approx 12 mL of the mononuclear cell volume should be obtained. Move the pipet tip in a circular, continuous manner over the mononuclear cell layer when aspirating avoiding red blood cell contamination. Discard the red blood cell contents in to the waste container containing 10% bleach solution.

9. Add 15 mL of PBS buffer to the mononuclear cell suspension, mix well by aspirating gently with the 10-mL pipet.

10. Place tubes with the mononuclear cell suspension in the centrifuge, spin for 18 min 350g at room temperature. Decant the supernatant, blot on a paper towel, resuspend in 6 mL of PBS buffer.

11. Deplete the cell suspension of monocytes and macrophages by incubation in 150-cm2 tissue culture flask for 2 h at 37°C (monocytes and macrophages will adhere to the surface of the flask). Wash off the nonadherent cells with PBS buffer and collect.

12. Optionally, remove the residual red blood cells by the addition of 25 mL of lysis buffer. Incubate at room temperature for 5 min and wash with PBS buffer by centrifugation.

13. Count the cells using a hemacytometer (or an automated cell counter, such as a Coulter counter, if available). Adjust the cell concentration to 10 x 106 per mL. Set aside two 1 x 106 cell aliquots for FACS controls: a cell auto-fluorescence control, and an irrelevant primary antibody-PE conjugate binding control. Optionally, add a T-cell control using anti CD3 antibody, a monocyte control using anti CD14 antibody, and a leukocyte control using anti CD45 antibody.

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