Transgenic and wild type FVB mice, born within the same litter, were euthanized by CO 2 inhalation. The bone marrow cells were flushed from femurs and tibias by using a 23-gauge needle with 10 mL of cold PBS. Single cell suspensions were prepared by flushing the cell solution twice through a 22-gauge needle and passing the cell solution through a sterile 40-mm nylon mesh. Spleens were removed aseptically, injected with 2 mg/mL of collagenase D and 20 mg/mL of DNase I (Roche Diagnostics, Mannheim, Germany) in Hanks solution, minced, incubated for 30 min at 37uC under 5% CO 2 , and teased through a 40 mm nylon mesh. Red blood cells in bone marrow cells and spleen cells were lysed twice using Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich, Saint Louis, MO 63103). Cells were washed twice with RPMI-1640 medium and subsequently suspended in RPMI-1640 culture medium containing 10% FBS and antibiotics. Cell viability was determined by trypan blue dye exclusion.