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.
Fondazione-Policlinico Tor Vergata, Rome, Italy.
Curr Protoc. 2023 Feb;3(2):e676. doi: 10.1002/cpz1.676.
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 ), intermediate (intM; CD14 CD16 ), and non-classical (ncM; CD14 CD16 ) 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.
单核细胞是先天免疫系统的关键成员，在感染和炎症过程中发挥关键作用。它们迁移到组织中并分化为髓样效应细胞（巨噬细胞、树突状细胞），这些细胞协调炎症过程，并充当先天免疫和适应性免疫反应之间的接口。它们在生理（妊娠）和病理（乳腺炎、子宫内膜炎和病毒感染）条件下对牛（Bos taurus）和水牛（Bubalus bubalis）这两种最重要的家畜的健康和疾病的临床相关性已得到强调。通过流式细胞术（FC）确定了牛存在三种不同的单核细胞亚群：经典（cM；CD14 CD16 ）、中间（intM；CD14 CD16 ）和非经典（ncM；CD14 CD16 ）单核细胞。FC 应用于研究牛和水牛的免疫系统仍然存在重大限制。在本文中，我们描述了一些实用方法来克服这些限制，特别是允许在牛和水牛外周血中识别和计数 cM、intM 和 ncM 亚群。事实上，我们提出了新的 lyse/wash/no-centrifugation（L/W/NC）程序，可以与 FC 绝对计数程序结合使用，并可以克服 lyse/no-wash 方案（L/NW）的特定问题。最后，我们首次证明了水牛中也存在 cM、intM 和 ncM 单核细胞亚群，这些亚群与牛有一些有趣的差异，例如水牛的 cM 主要是 CD14 /CD16 。这些细微的差异可能会影响例如乳腺炎和子宫内膜炎等炎症性疾病的调节。cM 上 CD16 表达的上调可能揭示了不同的单核细胞启动，导致感染后组织中巨噬细胞/树突状细胞的不同功能特征。 © 2023 Wiley Periodicals LLC. 基本方案：无补偿的 cM、intM 和 ncM 的绝对计数 替代方案：用于单激光平台的 cM、intM 和 ncM 的绝对计数 支持方案 1：使用 Pacific Blue 试剂盒进行内部单克隆抗体标记 支持方案 2：使用 Alexa Fluor 647 试剂盒进行内部单克隆抗体标记 支持方案 3：荧光标记抗体的滴定
