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Summary: Here, we present a protocol for the examination of immune cells in the murine conjunctiva and lacrimal gland using flow cytometry. We describe steps for dissection, preparation of high-quality single-cell suspensions, utilization of comprehensive staining panels, and optimization of flow cytometry voltage. We then detail procedures for compensation adjustments and the implementation of effective gating strategies.For complete details on the use and execution of this protocol, please refer to Ma et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.