100 μm mesh cell strainer

Mice were anesthetized (pentobarbital injection, i.p.) and livers were washed (with 50 ml of Krebs-HEPES, pH 7.65, supplemented with 0.5 mM EDTA) and digested (with 50 ml of Krebs-HEPES, pH 7.65, containing 25 mg of collagenase from Clostridium hystolyticum and 0.5 mM CaCl₂) by perfusion through the inferior vena cava at a rate of 5 ml min⁻¹ as described18 (link). The liver was removed and hepatocytes were extracted in attachment medium (DMEM supplemented with 1 g l⁻¹ glucose, 4 mM glutamine, 1 mM pyruvate, penicillin/streptomycin, 10% (v/v) FBS, 10 nM insulin, 200 nM triiodothyronine (T3) and 500 nM dexamethasone). After filtering through a 100-μm mesh cell strainer (BD Falcon), cells were pelleted (50g × 2 min) and resuspended in attachment medium for counting and seeding. Typically, cells were distributed in six-well plates, 2 ml per well containing 2.5 × 10⁵ cells. After attachment for 4 h, the cells were washed in PBS and incubated for 20 h in overnight medium (DMEM containing 1 g l⁻¹ glucose, 4 mM glutamine, 1 mM pyruvate, penicillin/streptomycin and 100 nM dexamethasone). Before treatment, the medium was replaced with fresh overnight medium. Unless otherwise stated, the cells were incubated first with 10 μM 991 (or 0.1% (v/v) DMSO, vehicle controls) for 20 min before treatment with the indicated concentrations of glucagon (or PBS, vehicle controls) for 15 min.

Mice were lethally anesthetized for lymphoid organ harvest. Brain, lymph nodes (deep cervical), and spleens were harvested after transcardial perfusion with phosphate buffered saline (PBS). Solid organs were mechanically homogenized in Roswell Park Memorial Institute (RPMI) medium + 10% FBS + 1% penicillin-streptomycin, and filtered through a 100-μm mesh cell strainer (BD Falcon). Red blood cells were lysed from spleen samples and washed with PBS. To isolate tumor-infiltrating lymphocytes (TILs), brains were harvested on post-implantation day 20–22. Brains were processed as described previously [66 ]. Briefly, brains were were treated with DNase I (Sigma) and collagenase type IV (BRAND) in RPMI with 1% FBS and dissociated using gentleMACS dissociator, filtered and resuspended in 5 mL 70% Percoll, layered below 30% Percoll and centrifuged at 2000 rpm for 20 mins at RT. Cell layer at the 30%/70% interface was collected and washed with PBS.