1. Acid-citrate dextrose-treated blood (450 ml) was obtained from donors.
2. PBMC were isolated by centrifugation over lymphocyte separation medium.
3. After three washes in phosphate-buffered saline (PBS), the PBMC were counted and cryopreserved in 90% fetal bovine serum and 10% dimethyl sulfoxide at 10⁷ cells/ml.
4. Thawed PBMC were mitogenically stimulated in the following medium: Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 μg of phytohemagglutinin (PHA) per ml, 10 ng of phorbol 12-myristate-13-acetate per ml, nonessential amino acids, 5 mM β-mercaptoethanol, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, 100 U of penicillin per ml, and 100 μg of streptomycin per ml.
5. Where indicated, 0.2 μM A23187 (a calcium ionophore) was substituted for PHA in the medium.
Schematic figure of a density gradient centrifugation

 
Method:
1. Carefully layer 35 mL of diluted cell suspension over 15 mL of Ficoll-Paque in a 50 mL conical tube.
2. Centrifuge at 400×g for 30–40 minutes at 20 °C in a swinging- bucket rotor without brake.
3. Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.
4. Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.
5. Fill the conical tube with buffer, mix, and centrifuge at 300×g for 10 minutes at 20 °C. Carefully remove supernatant completely.