Kandhi Rajani, Variya Bhavesh, Ramanathan Sheela, Ilangumaran Subburaj
Department of Immunology and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Québec, Canada.
Anat Rec (Hoboken). 2023 May;306(5):1011-1030. doi: 10.1002/ar.25039. Epub 2022 Jul 18.
Flow cytometry is an imperative tool to characterize alterations in a wide range of immune cell populations during inflammatory conditions and disease states that affect the liver such as the obesity-induced non-alcoholic fatty liver disease and liver fibrosis. Identification and quantification of immune cell subsets from the liver is critically dependent on efficient isolation of intrahepatic leukocytes. The isolation of leukocytes from fatty and fibrotic livers and processing the cells for flow cytometry can be challenging with respect to cell yields, purity and most importantly, the level of autofluorescence resulting from fat deposition. Here, we describe an efficient method for isolating intrahepatic leukocytes from mice fed with high fat diet and propose a strategy to alleviate autofluorescence during phenotyping by multicolor flowcytometry. We also describe a gating strategy for robust identification of granulocytes, pro-inflammatory, anti-inflammatory and transitional state monocyte subsets, dendritic cells, B cell, T lymphocyte subpopulations and NK cell subsets. Overall, the procedures described here will allow simultaneous processing of several samples while ensuring reproducible cell isolation and efficient noise reduction required for reliable characterization of intrahepatic leukocytes from the fatty liver tissues.
流式细胞术是一种重要工具，可用于表征在炎症状态和影响肝脏的疾病状态（如肥胖诱导的非酒精性脂肪性肝病和肝纤维化）期间多种免疫细胞群体的变化。从肝脏中鉴定和定量免疫细胞亚群严重依赖于肝内白细胞的有效分离。从脂肪性和纤维化肝脏中分离白细胞并对细胞进行流式细胞术处理，在细胞产量、纯度方面，最重要的是在脂肪沉积导致的自发荧光水平方面，可能具有挑战性。在这里，我们描述了一种从高脂饮食喂养的小鼠中分离肝内白细胞的有效方法，并提出了一种通过多色流式细胞术在表型分析过程中减轻自发荧光的策略。我们还描述了一种门控策略，用于可靠地鉴定粒细胞、促炎、抗炎和过渡状态单核细胞亚群、树突状细胞、B细胞、T淋巴细胞亚群和NK细胞亚群。总体而言，这里描述的程序将允许同时处理多个样本，同时确保可重复的细胞分离以及可靠表征脂肪肝组织中肝内白细胞所需的有效降噪。
