Boumelhem Badwi B, Assinder Stephen J, Bell-Anderson Kim S, Fraser Stuart T
a Discipline of Physiology , University of Sydney , Sydney , Australia.
b Bosch Institute, University of Sydney , Sydney , Australia.
Adipocyte. 2017 Apr 3;6(2):112-123. doi: 10.1080/21623945.2017.1319536. Epub 2017 Apr 14.
Understanding adipose tissue heterogeneity is hindered by the paucity of methods to analyze mature adipocytes at the single cell level. Here, we report a system for analyzing live adipocytes from different adipose depots in the adult mouse. Single cell suspensions of buoyant adipocytes were separated from the stromal vascular fraction and analyzed by flow cytometry. Compared to other lipophilic dyes, Nile Red uptake effectively distinguished adipocyte populations. Nile Red fluorescence increased with adipocyte size and granularity and could be combined with MitoTracker® Deep Red or fluorescent antibody labeling to further dissect adipose populations. Epicardial adipocytes exhibited the least mitochondrial membrane depolarization and highest fatty-acid translocase CD36 surface expression. In contrast, brown adipocytes showed low surface CD36 expression. Pregnancy resulted in reduced mitochondrial membrane depolarisation and increased CD36 surface expression in brown and epicardial adipocyte populations respectively. Our protocol revealed unreported heterogeneity between adipose depots and highlights the utility of flow cytometry for screening adipocytes at the single cell level.
由于缺乏在单细胞水平分析成熟脂肪细胞的方法，对脂肪组织异质性的理解受到阻碍。在此，我们报告了一种用于分析成年小鼠不同脂肪库中活脂肪细胞的系统。从基质血管部分分离出浮力脂肪细胞的单细胞悬液，并通过流式细胞术进行分析。与其他亲脂性染料相比，尼罗红摄取能有效区分脂肪细胞群体。尼罗红荧光随脂肪细胞大小和颗粒度增加，并且可以与MitoTracker® Deep Red或荧光抗体标记相结合，以进一步剖析脂肪群体。心外膜脂肪细胞表现出最低的线粒体膜去极化和最高的脂肪酸转运蛋白CD36表面表达。相比之下，棕色脂肪细胞表面CD36表达较低。妊娠分别导致棕色和心外膜脂肪细胞群体中线粒体膜去极化减少和CD36表面表达增加。我们的方案揭示了脂肪库之间未报道的异质性，并突出了流式细胞术在单细胞水平筛选脂肪细胞的实用性。
