Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

GOJANOVICH, ALDANA D.; GIMENEZ, MARÍA C.; MASONE, DIEGO; RODRIGUEZ, TANIA M.; DEWEY, RICARDO A.; DELGUI, LAURA R.; BUSTOS, DIEGO M.; UHART, MARINA

Frontiers in Cell and Developmental Biology

www.frontiersin.org

Lugar: Laussanne; Año: 2018 vol. 6

Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interestbecause of their potential for therapeutic approaches. The method described herecovers every single step necessary for hASCs isolation from subcutaneous abdominaladipose tissue, multicolor phenotyping by flow cytometry, and quantitative determinationof adipogenic differentiation status by means of lipid droplets (LDs) accumulation, andWestern blot analysis. Moreover, to simultaneously analyze both LDs accumulation andcellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO)staining with immunofluorescence detection. For LDs quantification we wrote a programfor automatic ORO-stained digital image processing implemented in Octave, a freelyavailable software package. Our method is based on the use of the traditional low costneutral lipids dye ORO, which can be imaged both by bright-field and fluorescencemicroscopy. The utilization of ORO instead of other more expensive lipid-specificdyes, together with the fact that the whole method has been designed employingcost-effective culture reagents (standard culture medium and serum), makes it affordablefor tight-budget research laboratories. These may be replaced, if necessary or desired,by defined xeno-free reagents for clinical research and applications.