Summary: Here, we present a protocol for collecting, dissociating, isolating, staining, and analyzing immune cells from pancreatic cancer tissues for flow cytometry. The isolated cells can also be used for single-cell RNA sequencing and other related procedures.For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.