Flow Cytometry of Hematological and Immunological Samples from Different Species

Szalóki, Gábor; Czeti, Ágnes

doi:10.1002/cyto.a.24222

Cited by 1 publication

(1 citation statement)

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“…However, the single‐platform method should be considered the gold standard for leukocyte subpopulation absolute counts, as shown by the European Working Group on Clinical Cell Analysis (EWGCCA; Gratama et al., 1998). Therefore, the use of L/NW staining protocols is highly recommended accordingly (Brando et al., 2000; Szaloki & Czeti, 2021).…”

Section: Commentarymentioning

confidence: 99%

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood

et al. 2023

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Monocytes 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 compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In‐house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In‐house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome‐conjugated antibodies

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Section: Commentarymentioning

confidence: 99%