Analysis and Isolation of Adipocytes by Flow Cytometry

Abstract

Analysis and isolation of adipocytes via flow cytometry is particularly useful to study their biology. However, the adoption of this technology has often been hampered by the presence of stromal/vascular cells in adipocyte fractions prepared from collagenase-digested adipose tissue. Here, we describe a multistep staining method and gating strategy that effectively excludes stromal contaminants. Initially, we set a gate optimized to the size and internal complexity of adipocytes. Exclusion of cell aggregates is then performed based on fluorescence of a nuclear stain followed by positive selection to collect only those cell events containing lipid droplets. Lastly, negative selection of cells expressing stromal or vascular lineage markers removes any remaining stromal contaminants. These procedures are applicable to simple analysis of adipocytes and their subcellular constituents by flow cytometry as well as isolation of adipocytes by flow sorting.

Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression

“…35 One problem associated with the present study and previous studies relates to the presence of cells from the stromal vascular fraction, including immune cells, in the isolated mature adipocytes. While elaborate FACS-based protocols minimizing the presence of cells from the stromal vascular fraction in the fraction representing the mature adipocytes have been developed, 36 they still do not guarantee the complete absence of cells from the stromal vascular fraction. To completely rule out such contamination, analyses based on single isolated adipocytes would seem to be required.…”

Epigenome-wide profiling of DNA methylation in paired samples of adipose tissue and blood

“…This affords the unique opportunity to interrogate blood to define the profile of immune cells in any normal or disease individual using DNA methylation rather than flow cytometry, for example. Cell sorting in solid tissue or finer cellular phenotyping 42 will provide better insight into the nature and importance of individual tissue heterogeneity.…”

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

“…Cytological analysis with labelled miRNA mimics however is limited by the inaccuracy of fluorescence microscopy in detecting miRNA behaviour [14]. This lack of accuracy is due to the low number of detected cells, with approximately 200-400 cells detected per field, though fluorescence microscopy is more accurate and quantitative in single cells [15]. Thus during key cellular events, subcellular information reflecting the cell population of interest is typically not accurate [16].…”

“…The ability to perform photometric or morphometric analysis of the images of thousands of individual cells thus combines quantitative image analysis with the statistical power of flow cytometry [15]. This technology monitors miRNA behaviour using a fluorescence tag in a high-throughput approach that is amenable to statistical analysis.…”