Figure 2.

Multistep flow cytometry/sorting strategy to purify human adipocytes. A) Gating strategy for adipocyte isolation is diagramed from top to bottom. In the first step, adipocytes are identified by their large size and refractile properties in a plot of FSC vs. SSC. Singlets were identified optically by comparing SSC peak height to peak width. In the third step, LipidTOX fluorescence of the singlets identified lipid droplet-containing cells and allowed exclusion of dead cells and free nuclei based on DAPI fluorescence. Only lipid^POS/DAPI^NEG cells were collected. Finally, any remaining stromal contaminants are excluded based on their labeling with FITC-conjugated antibodies to CD45. PE, phycoerythrin channel. B) Immediately following flow sorting of the adipocytes, their purity was checked by flow cytometry analysis of a small portion of the sorted cells. All sorted cells were intact and alive (DAPI^NEG), and CD45^NEG. C) RT-PCR was used to verify the purification strategy. The results show the presence of adipocyte markers (AdipoQ and Plin-1) in purified adipocytes and whole-adipose tissue homogenate. However, hematopoietic (CD45 and CD11b) and mesenchymal (PDGFRα and Flk1) markers were only detected in the tissue homogenate and essentially undetectable in flow-purified adipocytes. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.