Immune Profiling of the Tumor Microenvironment

For ELISpot and intracellular cytokine staining (ICS) by flow cytometry, spleens were collected and crushed through a 70μM cell strainer to form a single cell suspension. Red blood cells were lysed (Tonbo Biosciences, San Diego, CA) and splenocytes were counted (Cellometer Auto 2000, Nexcelom Bioscience, Lawrence MA). To analyze the TME, mice were treated with brefeldin A, as previously described (21 (link)) (Sigma-Aldrich, St. Louis, MO, 250μg per mouse) for four to six hours before euthanization. Tumors were collected, weighed, processed (Tumor Dissociation Kit and gentleMACS dissociator, Miltenyi Biotec, Germany), and total cells were counted.