Flow Cytometry Strategies for Rapidly Characterizing Heterogeneous Adipocyte Populations in 3D In Vitro Constructs

Adipose tissue (AT) is an endocrine organ that regulates whole body metabolism and supports energy needs of other tissues. Two key adipose tissue functions are insulin-stimulated glucose uptake and lipid metabolism. As the prevalence of metabolic diseases, such as obesity, continue to rise, there is a growing need for new methods to study adipose tissue and its main cell type, adipocytes. Adipocytes are unique cells, distinguished by their large spherical shape housing large lipid droplet(s). For many in vitro models (and in tissues), adipocytes are derived from a heterogenous population of precursor cells, leading to varying degrees of adipogenesis and adipocyte maturation. Characterization of such populations can be challenging because often the average result does not account for the complexity of the various sub-populations. Common single cell characterization methods provide data based gene and protein expression but do not account for the morphological variability in cells such as adipocytes at different stages of maturation, and are expensive to run. More traditional methods, such as microscopy or colorimetric assays, are often time consuming with intrinsic challenges due to overlapping or coincident features or lose single cell adipocyte details due to the destructive nature of the assay. Here, we show how flow cytometry can be used to characterize adipocyte populations while preserving critical details at the individual adipocyte level. This protocol provides multiple workflows for indirect measurements of lipogenesis (lipid accumulation), protein content (branched actin formation), and adipocyte functions (insulin-stimulated glucose uptake). These flow cytometry workflows show the effectiveness of binning individual adipocytes based on their level of maturity and allow for comparisons within subpopulations traditional methods cannot provide.