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Independent Measurements of Lipids in Mixed Cell Populations
Author(s) -
Stoeckman Angela,
Estrada Katerina,
Wolins Nathan
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.629.1
Subject(s) - lipid droplet , organelle , lipid metabolism , cell , lipidomics , fatty liver , cell type , adipocyte , biology , cholesterol , flow cytometry , triglyceride , chemistry , adipose tissue , biochemistry , microbiology and biotechnology , disease , pathology , immunology , medicine
Metabolic diseases such as type 2 diabetes, atherosclerosis, and non‐alcoholic fatty liver disease (NAFLD) are a major cause for concern worldwide with one in three being diagnosed with NAFLD in the United States. NAFLD is a better indicator of metabolic disease than obesity and it is likely that how fat is deposited at the cellular level, rather than the organ level, will be an even better predictor of metabolic disease than obesity or NAFLD alone. In each of the aforementioned metabolic diseases, there is a pathological increase in the amount of lipid stored in specific cell types such as adipocytes, myocytes, macrophages, or hepatocytes leaving the cell or tissue vulnerable to a multitude of cellular stresses. The stored lipids are predominantly triglycerides and cholesterol esters and are sequestered into organelles called lipid droplets. These organelles have the dynamic ability to change in size based on the needs of the organism. Therefore, the ability to accurately assess changes in the amount of lipid in these droplets is essential. Traditionally, methods to quantify lipids have included cell isolation and lysis followed by organic lipid extraction and measurement of triglyceride mass or cell microscopy techniques where lipids are visualized through the use of fluorescent stains. These current techniques are time‐consuming and cannot differentiate between unique lipid levels within the same sample. Here, we demonstrate the use of a lipophilic fluorochrome in a flow cytometry assay as a valid method for quantifying lipid droplets in multiple cell types as a direct comparison against traditional methods. We observed a dose‐dependent increase in mean fluorescence intensity in multiple cell types treated with the unsaturated fatty acid oleate. There was a positive correlation between our flow cytometry technique and the triglyceride assay and fluorescence microscopy techniques. The advantage to our flow cytometry‐based approach was improved sensitivity obtained in a fraction of the time as compared to traditional methods. Also, we highlight the ability of our method to assess differing levels of lipid droplets within a mixed cell population of AML‐12 mouse hepatocytes and 293 human kidney cells fed different amounts of oleate as well as within human peripheral blood mononuclear cells. Further, when gating only on lymphocytes within a mixture of human peripheral blood mononuclear cells, subpopulations could be resolved by lipid levels. Using flow cytometry for the quantitation of lipid droplets will allow researchers and clinicians to obtain accurate data quickly, reproducibly, and within human samples which may contain a mixed population of cells each with distinct levels of lipid stores.