Gojanovich Aldana D, Gimenez María C, Masone Diego, Rodriguez Tania M, Dewey Ricardo A, Delgui Laura R, Bustos Diego M, Uhart Marina
Laboratorio de Integración de Señales Celulares, IHEM, Universidad Nacional de Cuyo, CONICET, Mendoza, Argentina.
Facultad de de Ciencias Veterinarias y Ambientales, Universidad Juan Agustín Maza, Mendoza, Argentina.
Front Cell Dev Biol. 2018 Apr 4;6:33. doi: 10.3389/fcell.2018.00033. eCollection 2018.
Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.
人脂肪来源的间充质干/基质细胞（hASCs）因其在治疗方法中的潜力而备受关注。本文所述方法涵盖了从腹部皮下脂肪组织中分离hASCs、通过流式细胞术进行多色表型分析、通过脂滴（LDs）积累和蛋白质印迹分析定量测定成脂分化状态所需的每一个步骤。此外，为了通过荧光显微镜同时分析LDs积累和细胞蛋白定位，我们将油红O（ORO）染色与免疫荧光检测相结合。对于LDs定量，我们编写了一个用于自动ORO染色数字图像处理的程序，该程序在免费软件包Octave中实现。我们的方法基于使用传统的低成本中性脂质染料ORO，它可以通过明场和荧光显微镜成像。使用ORO代替其他更昂贵的脂质特异性染料，以及整个方法采用具有成本效益的培养试剂（标准培养基和血清）设计这一事实，使得预算紧张的研究实验室也能够负担得起。如有必要或需要，这些试剂可被用于临床研究和应用的无动物源试剂所替代。
