Affiliation:
1. CREA‐Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria Centro di ricerca Zootecnia e Acquacoltura (Research Center for Animal Production and Aquaculture) Monterotondo Italy
2. Fondazione‐Policlinico Tor Vergata Rome Italy
3. Department of Biomolecular Sciences University of Urbino “Carlo Bo” Urbino Italy
4. Department of Biomedicine and Prevention Tor Vergata University Rome Italy
5. Institute for Immunology University of Veterinary Medicine Foundation Hanover Germany
6. Department of Microbiology, College of Veterinary Medicine King Faisal University Al‐Ahsa Saudi Arabia
Abstract
AbstractMonocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions.The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++CD16–/low), intermediate (intM; CD14++/+CD16+), and non‐classical (ncM; CD14–/lowCD16++) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no‐centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no‐wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++/CD16+. These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC.Basic Protocol: Absolute count of cM, intM, and ncM without compensationAlternate Protocol: Absolute count of cM, intM, and ncM for single laser platformSupport Protocol 1: In‐house monoclonal antibody labeling using a Pacific Blue™ kitSupport Protocol 2: In‐house monoclonal antibody labeling using an Alexa Fluor® 647 kitSupport Protocol 3: Titration of fluorochrome‐conjugated antibodies
Subject
Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience