Neutral lipid droplets (LDs) are dynamic lipid storage organelles found in all eukaryotic cells from yeast to mammals and higher plants. LDs are important to many physiological processes that include basic cellular maintenance, metabolism, and diverse medical pathologies. LD accumulation has been studied extensively by a range of methods, but particularly by microscopy with several fluorescent dyes extensively used for qualitative and quantitative imaging. Here, we compared established LD stains Nile Red and BODIPY 493/503 to the 4', 6-diamidino-2-phenylindole (DAPI)-range dye 1,6-diphenyl-1,3,5-hexatriene (DPH; excitation/emission λmax=350 nm/420 nm) using high-content image analysis. HeLa cells treated with oleic acid or vehicle were used to compare staining patterns between DPH and Nile Red as well as DPH and the LD protein adipophilin. DPH, Nile Red, and BODIPY 493/503 were compared as assay reagents in oleic acid dose-response experiments. Treatment of MCF-7 cells with sodium butyrate was used as a second cellular system for high-content analysis of LD formation. In this experimental context, we demonstrate the compatibility of DPH with GFP, a technical limitation of Nile Red and BODIPY 493/503 dyes. These data show that DPH has comparable sensitivity and specificity to that of Nile Red. Z'-factor analysis of dose-response experiments indicated that DPH and BODIPY 493/503 are well suited for quantitative analysis of LDs for high-throughput screening (HTS) applications.
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