Protocol for Dissociation and Isolation of Murine Tumor and Brain Cells

(A) Mouse pancreatic tumors were washed in RPMI 1640 (Gibco, Life Technologies) and cut into 2-4mm pieces immediately following resection. Dissociation into a single cell suspension was performed using the Miltenyi Biotec Mouse Tumor Dissociation Kit (130-096-730). Briefly, tumor pieces were collected into gentleMACS C tubes containing RPMI 1640 dissociation enzymes, and further homogenized using the gentleMACS Dissociator. Samples were incubated for 40 minutes at 37°C under continuous rotation, then passaged through a 70 μm nylon mesh (Corning). Red blood cells were lysed using RBC Lysis Buffer (eBioscience), and the remaining tumor cells were used for FACS analysis and cell sorting. (B) Freshly resected mouse brains were rinsed in PBS, placed in accutase (Life Technologies), and cut into <2mm pieces. Samples were incubated 15 minutes at 37°C, then passaged through a 70 μm nylon mesh (Corning). Red blood cells were lysed as above prior to FACS analysis and sorting of brain cells. (C) Bone marrow cells were suspended in HBSS (Gibco, Life Technologies) containing 5% FBS and 2 mM EDTA and were prepared for FACS analysis and sorting as previously described^{31 (link)}. Analysis and cell sorting were carried out on a FACSAria III machine (Becton Dickinson), and data were analyzed with FlowJo software (Tree Star).