Fresh peripheral blood was diluted with an equal amount of phosphate buffered saline (PBS) and was then slowly transferred to a 15-ml glass tube containing lymphocyte separation medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).
The tissue preparation was then suspended in 50 ml KBM-400 (Kojin Bio Co., Tokyo, Japan) or Alyse (ALyS505N: Cell Science and Technology Institute, Inc., Sendai, Japan) serum-free lymphocyte medium containing 400 IU/ml human recombinant interleukin 2 (Proleukin; Chiron B.V., Amsterdam, Netherlands), transferred to a 75-cm culture flask, and incubated at 37 °C in air containing 5%% CO2.
The corresponding inactivated antigen was added to a 100 μl volume of medium containing 2 × 10 lymphocytes, performed in triplicate in a 96-well plate format.
Human lymphocyte cell line (ATCC PCS-800-013) was cultured in RPMI medium containing 10% fetal bovine serum, antibiotics, and phytohemagglutinin M. The human lymphocyte cultures were incubated in 5% CO2 incubator at 37°C.
The cystic wall was thickened and the cystic fluid was yellowish serous with containing lymphocytes.
After exposure, lymphocytes were incubated in growth medium containing phytohemagglutinin (PHA) and chromosome spreads were collected using a chemical-induced premature chromosome condensation (PCC) technique.
Re-stimulated lymphocytes were cultured in RPMI-HEPES medium containing 10% FCS, 2 m M glutamine, combined antibiotics, 1 m M sodium pyruvate, 25 m M HEPES, 5 × 10−5 M βME, and 1% NEAA for 5 days.
CD8+ T lymphocytes were then cultured in RPMI1640 medium containing 10% human serum, anti-CD3 monoclonal antibody (OKT3), interleukins (IL -2, IL -2 IL-75 and IL-15 for 3 dand.
Restimulated lymphocytes were cultured in RPMI-HEPES medium containing 10% FCS, 2 m M glutamine, combined antibiotics, 1 m M sodium pyruvate, 25 m M HEPES, 5 × 10−5 M βME and 1% NEAA for 5 days.
Isolated B Lymphocytes were cultured (10 cells/ml) with complete RPMI-1640 medium containing 15% fetal calf serum + L-glutamine 2 mM, HEPES 1 mM, penicillin 100 U/mL, streptomycin 100 mg/mL in 24-well plates at 37°C.
