Orr Jeb S, Kennedy Arion J, Hasty Alyssa H
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, USA.
J Vis Exp. 2013 May 22(75):e50707. doi: 10.3791/50707.
The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.
在肥胖啮齿动物和人类的脂肪组织（AT）中发现巨噬细胞浸润增加，这引发了人们对免疫细胞在局部和全身胰岛素抵抗中作用的兴趣激增。在免疫代谢实验室中，分离和定量瘦人和肥胖者AT中的不同免疫细胞群体是一种常用技术；然而，在基质血管细胞分离和流式细胞术分析过程中都必须格外小心，以便获得可靠且可解释的数据。在本视频中，我们展示了如何切碎、消化并分离富含免疫细胞的基质血管部分。随后，我们展示了如何用抗体标记巨噬细胞和T淋巴细胞，以及如何在流式细胞术实验中对它们进行正确的设门。提供了来自低脂喂养的瘦小鼠和高脂喂养的肥胖小鼠的代表性流式细胞术图。该分析的一个关键要素是使用在AT巨噬细胞自然自发荧光的通道中不发荧光的抗体，以及使用适当的补偿对照。
