Background

Microglia are brain-resident immune cells that contribute to the neuroenvironment through diverse functions, including debris phagocytosis, neuronal support, and synaptic pruning. Their multifaceted roles align with distinct phenotypic states, largely influenced by environmental stimuli and intrinsic signaling cascades [1]. These specialized cells are involved in many neurological diseases, such as Alzheimer’s disease (AD). For instance, the disease-associated microglia (DAM) phenotype was first identified in an AD mouse model and has since been further validated to play a role in response to neurodegeneration [2]. Differentiating and quantifying microglial phenotypic states offers valuable insights into pathways associated with development and disease processes, presenting promising avenues for therapeutic exploration. Although fluorescent immunolabeling and microscopy can be used to distinguish microglial phenotypes, this approach is time-consuming, and quantification requires multiple tissue sections. Alternatively, sequencing-based approaches, though providing high-throughput and high-resolution data, are costly and demand specialized analytical training. Here, we present a fluorescence-based flow cytometry method to distinguish microglial phenotypic states, allowing for high-throughput, multiparametric analysis of single-cell suspensions with straightforward quantification tools.

In this protocol, we describe a simple and time-effective preparation of single-cell suspensions from murine brain tissue for flow cytometric analysis to distinguish multiple microglial phenotypes. Microglia have been previously described as either “M1” (pro-inflammatory) or “M2” (anti-inflammatory) to indicate “active” or “resting” states, respectively. However, the field has since moved to more descriptive terms such as “homeostatic” or specific “reactive” phenotypes, given that microglia are in a constant state of activity to respond to stimuli [3]. Therefore, this panel can distinguish between non-reactive/surveying (homeostatic) or reactive [DAM, interferon-response microglia (IRM), and lipid-droplet accumulating microglia (LDAM)] microglial phenotypes [3]. This protocol was previously used to examine sex differences in microglial phenotypic states during aging [4]. The markers for each of the examined phenotypes (homeostatic, DAM, IRM, and LDAM) were selected from several publications [2,5–10], ensuring no overlap between states, and are listed within the Materials and Reagents section.

A notable advantage to this protocol is the adaptability of this microglial phenotype panel, allowing for additional fluorescent antibodies or dyes depending on the laser and filter capabilities of the chosen cell analyzer. The use of fluorescent dyes offers in vivo examination of microglial phenotypes, including Methoxy-X04 (amyloid-β plaques) [11] and BODIPY (lipid accumulations) [10], where colocalization with these markers is useful in identifying DAM and LDAM phenotypes, respectively. This flexibility facilitates versatile applications in studying microglia. While initially utilized to examine sex differences in hippocampal microglial aging, this protocol can be applied to address various biological questions relevant to multiple murine disease models and other brain regions.