Protocol for optimized mononuclear cell isolation from liver and tumor tissue using mechanical or enzymatic digestion

Mononuclear cells from human liver tissue, including tumor and non-tumor regions, are a valuable source for studies of the tumor microenvironment. Here, we present a protocol to dissociate liver tissue from patients with hepatocellular carcinoma. We provide steps to generate single-cell suspension using either mechanical or enzymatic digestion, following steps for separating mononuclear cells using density gradient. Finally, we describe steps for flow cytometry staining of isolated immune cells. For complete details on the use and execution of this protocol, please refer to Moreno-Fernandez et al.¹ and Heinrich et al.².