Isolation and Purification of Lymphocytes from Murine Tissues

Mice were sacrificed by isofluorane treatment. Cardiac puncture was performed prior to the harvesting of the organs. Livers were perfused with PBS-Serum (PBS +1% Fetal Bovine Serum) before harvesting. Spleens were dissociated using a plunger from a 3 mL syringe and lymphocytes were enriched using Lympholyte Cell Separation Media (Accurate Chemical). Livers were dissociated using a gentleMACS Dissociator (Miltenyi Biotec) and lymphocytes were enriched using a 40–70% discontinuous Percoll gradient (GE Healthcare) as previously described [14] (link). Salivary gland lymphocytes were prepared as described [14] (link). Briefly, SMGs were removed of all lymph nodes and connective tissue, followed by mincing. Single cell dissociation was performed using one incubation with digestion medium (RPMI 1640 containing 1 mg/ml of collagenase IV (Sigma), 5 mM CaCl2 50 µg of DNase I (Sigma) and 8% FBS) and continuous shaking at room temperature. The digestion mixture was pipetted vigorously to dissociate remaining cells. Supernatant was collected, passed through nylon mesh and the lymphocytes purified by layering on a lympholyte-M gradient. Bone marrow cells were isolated from femur and tibia. Red blood cells were lysed with ammonium chloride lysis buffer.