Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle, Germany.
Cytometry A. 2010 Oct;77(10):990-5. doi: 10.1002/cyto.a.20953. Epub 2010 Aug 31.
Because of the increasing incidence of worldwide obesity, cell culture models which enable the study of adipose tissue development are of particular importance. The murine embryonic stem cell (ESC) line CGR8 differentiates into adipocytes with a differentiation efficiency of up to 15%. A critical step for the analysis of stem cell-derived adipogenesis is the reliable separation of adipocytes. Here we report on how to (i) gently separate the cells of embryoid bodies (EBs) and (ii) identify and sort adipocytes from the rest of the heterogeneous cell mixture. Up to the present, no adipocyte specific surface marker is known for fluorescence activated cell sorting (FACS). After separation we employed two independently existing FACS methods for adipocyte cell sorting. These methods are based on Nile red staining and granularity. For stem cell-derived adipocytes only the combination of both methods led to a reliable, efficient, and highly reproducible FACS analysis, as shown by the presence and absence of adipocyte specific markers in positively and negatively sorted cells.
由于全球肥胖症发病率的不断上升，能够研究脂肪组织发育的细胞培养模型显得尤为重要。鼠胚胎干细胞（ESC）系 CGR8 分化为脂肪细胞的分化效率高达 15%。分析干细胞衍生的脂肪生成的关键步骤是可靠地分离脂肪细胞。在这里，我们报告如何（i）温和地分离胚状体（EB）的细胞，以及（ii）从异质细胞混合物中鉴定和分选脂肪细胞。到目前为止，还没有用于荧光激活细胞分选（FACS）的脂肪细胞特异性表面标记物。分离后，我们采用了两种独立存在的基于尼罗红染色和粒度的 FACS 方法来分选脂肪细胞。对于源自干细胞的脂肪细胞，只有这两种方法的组合才能导致可靠、高效和高度可重复的 FACS 分析，这可以通过阳性和阴性分选细胞中存在和不存在脂肪细胞特异性标记物来证明。
