High-Dimensional Immune Profiling of Human Retinal Detachment Samples Using Spectral Flow Cytometry: A Protocol for Intraocular Immunotyping

Retinal detachment (RD) disrupts the eye's immune-privileged status, causing a local inflammatory response that contributes to adverse clinical outcomes, including proliferative vitreoretinopathy and suboptimal visual recovery. Comprehensive profiling of intraocular immune cells will offer mechanistic insights and support the development of personalized immunomodulatory strategies. Here, we describe a robust and standardized protocol for the collection and high-dimensional analysis of the intraocular immune infiltrate from patients undergoing RD surgery, using state-of-the-art spectral cytometry. Vitreous and retinal tissue samples were obtained during standard surgical procedures, without the need for additional invasive interventions. Our approach integrates two complementary protocols: one that enables selective isolation of immune cells by sorting for CD45⁺ populations, and a second one that applies a 39-color spectral cytometry panel to profile the general landscape of immune subpopulations. The panel can identify up to 62 distinct viable immune subsets per sample, along with their functional status, as it includes expression of 13 functional markers. Hence, we hereby detail sample preparation, staining, and acquisition workflow, as well as the gating strategy and essential steps necessary for reproducible immunophenotyping. Our protocol, which enables high-dimensional immune profiling from minimal biological material, provides a valuable platform for studying ocular inflammation in RD and other retinal diseases.