Isolation and Characterization of Immune Cells

Lymphoid and non-lymphoid organ DCs were harvested and prepared as described previously^{30 (link)}. Briefly, spleens and inguinal skin-draining LNs were minced and digested in 5 ml of IMDM +10% FCS (cIMDM) with 250 μg/ml of collagenase B (Roche) and 30 U/ml of DNaseI (Sigma-Aldrich) for 45 min at 37 °C with stirring. Lungs were minced and digested in 5 ml of cIMDM with 4 mg/ml of collagenase D (Roche) and 30 U/ml of DNaseI (Sigma-Aldrich) for 1.5 h at 37 °C with stirring. Tumours were minced and digested in serum-free IMDM with 125 μg/ml Liberase (Roche) and 30 U/mL of DNaseI (Sigma-Aldrich) for 45 minutes at 37 °C with stirring. After digestion was complete, single-cell suspensions from all organs were passed through 70-μm strainers and red blood cells were lysed with ammonium chloride–potassium bicarbonate (ACK) lysis buffer. Cells were subsequently counted with a Vi-CELL analyzer (Beckman Coulter) and 3–5×10⁶ cells were used per antibody staining reaction.