Eosinophil Essentials: Identification and Assessment of Activity.

Eosinophils are highly granulated white blood cells first identified as "granule blood cells" in the 19th century. In this review, we discuss techniques used to identify these unique cells and explore how they release a range of pro- and anti-inflammatory mediators. They are easily detected with a range of cellular dyes due to highly cationic proteins stored within their crystalloid granules. Cationic proteins include major basic protein (MBP), eosinophil peroxidase (EPX), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and Charcot-Leyden crystal protein (CLC, also known as galectin-10) that serve as potent antimicrobial factors and possess a high affinity for negatively charged molecules including eosin and fluorescein isothiocyanate (FITC). Crystalloid granules are responsible for autofluorescence and nonspecific binding to both fluorophores and antibodies during immunolabeling for fluorescence microscopy, flow cytometry, CyTOF, and other antibody-based detection methods. Eosinophils release a plethora of mediators that have roles in immunity and homeostasis. Here, we describe six different categories of mediators released by eosinophils and their analyses: (1) cationic granule proteins (MBP, EPX, EDN, ECP, and CLC), (2) cytokines and chemokines, (3) reactive oxygen species, (4) eicosanoid production, (5) eosinophil extracellular trap formation, and (6) exosomes. We also describe novel transcriptional markers where new subtypes of eosinophils are characterized through the development of single cell RNA sequencing, showing additional transcripts appearing in eosinophils from patients with diseases. Future work on eosinophils is anticipated to lead to a greater understanding of their role in immunity and diseases based on novel emerging techniques.