Anti mouse cd45 magnetic beads

Three weeks after injection of 3LL cells to C57BL/6 mice, the mice were killed and the lungs were perfused by injection of saline to the right heart ventricle. Next, the lungs were harvested and the left lobe tumors were carefully dissected from the left lung parenchyma. The right lung lobes were used as a non-tumor-bearing control sample. In each experiment, the tumor and lung samples from five mice were pooled together to yield a large number of leukocytes. Both lung and tumor specimens were roughly minced and put in RPMI containing collagenase IV and DNase (200 μg/ml and 5 μg/ml, respectively, both from Worthington, Lakewood, NJ) for 40 minutes at 37°C. Afterward, samples were meshed through a 70-μm disposable mesh (Falcon, BD Bioscience) and washed twice with phosphate buffered saline (wash buffer) containing 4% FBS (Biological Industries). Samples were reconstituted in 100-μl wash buffer, blocked with 1% mouse serum, and separated using anti-mouse CD45 magnetic beads (Miltenyi, Germany) as described by the manufacturer.