Multi-color Flow Cytometry Protocol to Characterize Myeloid Cells in Mouse Retina Research

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry–based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers.Key features• This protocol describes a flow cytometry–based method to analyze the myeloid cell response in retinopathy mouse models.• The protocol can distinguish between microglia- and monocyte-derived macrophages.• It can be modified to incorporate markers of interest.We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.