Human neonatal leukocytes were isolated from freshly collected, heparinized, neonatal cord blood by a density gradient centrifugation using Lymphoprep. Th2-polarized cells were generated by stimulating cord blood leukocytes with 200 U/ml IL-4 and 2 ug/ml neutralizing anti-IL-12 antibody for Th2 cultures. Cells were washed on day 3 and expanded in each medium containing 4 ng/ml IL-2. At day 12 to 14, leukocytes were incubated with anti-CD4 monoclonal antibody coated magnetic beads, and CD4+ cells were isolated by passing the cultured cells through a magnetic cell separation system. Cord blood-derived Th2 cells were collected after 3 days of CD4 positive cell separation, washed, and restumulated with 50 ng/ml of PMA and 1 ug/ml of ionomycin for 4 h.
