B16F10-luc2-tdTomato cells (1 × 106) were injected into the tail vein. After 24 hours, the lung was perfused, resected, minced, and digested in a collagenase solution (Collagenase II, 2 mg/ml, Sigma-Aldrich; deoxyribonuclease I, 40 μg/ml, Roche) for 45 min at 37°C. The digested tissue was passed through a 40-μm cell strainer before erythrocyte lysis with Pharm Lyse (BD). After washing and a second filtration step, the cell suspension was labeled with CD45-APC/Cy7 (103116, BioLegend). 7-AAD (7-aminoactinomycin D; 420404, BioLegend) was used for life/dead discrimination. CD45-negative, tdTomato-positive cells were counted as tumor cells homed into the lungs.

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