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Shan Yang, Wendy Kam, David A Sullivan, Yang Liu; Analysis of staining methods to identify neutral lipid expression in human meibomian gland epithelial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):126.
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© ARVO (1962-2015); The Authors (2016-present)
A critically important step in the differentiation of human meibomian gland epithelial cells (HMGECs) is the intravesicular accumulation of neutral lipids. However, the optimal method to identify such lipids is unclear. The goal of this study was to compare the utility of four different dyes for neutral lipid detection in immortalized (I) HMGECs.
IHMGECs were cultured in a FCS-containing medium for 10 days in the presence or absence of Roxadustat (Roxa), a known-inducer of IHMGEC differentiation (n = 2 wells/treatment/experiment; n = 3 experiments/dye). Cells were then fixed and stained with Oil Red O (ORO), Sudan III (SIII), LipidTox green (LT) or Nile Red (NR) and examined with an Eclipse E800 fluorescent microscope. IHMGECs were evaluated for the number, size and area of stained intracellular lipid vesicles, or the intensity of staining by using bright fields (ORO, SIII) or fluorescence (LT, NR). Data were captured with ImageJ and analyzed with Student’s two-tailed t test.
Our findings demonstrated that different staining methods can yield significantly different patterns of neutral lipid quantity and/or distribution in IHMGECs. ORO and SIII both demonstrated a significant increase in the size and area of neutral lipid-containing vesicles in Roxa-treated cells by using bright field microscopy. Neither stain showed a change in the number of vesicles during IHMGEC differentiation, but this may have been due to the apparent merging of vesicles within the cells. Vesicle size was often significantly greater in cells stained with ORO, as compared to SIII, and lipid visualization with the latter stain was limited by the propensity of SIII to precipitate. In contrast, it was not possible to evaluate the number, size or area of neutral lipid vesicles in IHMGECs with LT or NR, because of heightened background fluorescence. However, LT, but not NR, was able to show a significant increase in neutral lipid intensity in IHMGECs following Roxa-induced differentiation.
Overall, our results demonstrate that significant differences exist in the distribution patterns and intensities of neutral lipid-containing vesicles in IHMGECs after staining with ORO, SIII, LT and NR. ORO staining appeared to be the most useful method to help identify and quantitate the extent of neutral lipid accumulation during IHMGEC differentiation.
This is a 2020 ARVO Annual Meeting abstract.
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