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Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays.

Authors
  • Phan, Minh Anh Thu1
  • Madigan, Michele C2
  • Willcox, Mark2
  • Stapleton, Fiona2
  • Golebiowski, Blanka2
  • 1 School of Optometry and Vision Science, Faculty of Medicine and Health, UNSW, Sydney, NSW, 2033, Australia. Electronic address: [email protected] , (Australia)
  • 2 School of Optometry and Vision Science, Faculty of Medicine and Health, UNSW, Sydney, NSW, 2033, Australia. , (Australia)
Type
Published Article
Journal
Experimental Eye Research
Publisher
Elsevier
Publication Date
Sep 01, 2021
Volume
210
Pages
108719–108719
Identifiers
DOI: 10.1016/j.exer.2021.108719
PMID: 34364889
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Two spectrophotometric microplate assays with dual staining for either fluorescent Nile red (NR) plus 4,6-diamidino-2-phenylindole (DAPI) or non-fluorescent Oil red O (ORO) plus Crystal violet (CV) were applied and optimised to evaluate the lipid producing capacity of immortalised human meibomian gland epithelial cells (iHMGEC). Cells were treated with rosiglitazone (Rosi, 10-50 μM), a known lipid producing inducer for iHMGEC, and were analysed for lipids using the NR-DAPI and ORO-CV microplate assays. The lipid producing capacity of iHMGEC after each treatment was determined by normalising lipid quantity (measured with NR or ORO) to cell number (measured with DAPI or CV). The dye concentrations of NR 1 μg/mL, DAPI 5 μg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v), provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence). Both NR-DAPI and ORO-CV showed a dose-dependent effect of Rosi on lipid production in iHMGEC, consistent with the results reported previously using traditional microscopic imaging methods. The microplate assays offer a rapid, high throughput and objective measurement of the amount of lipids in iHMGEC (and potentially other lipid-producing cells) and can be used for screening the effects of biological agents or incubation changes on lipid production in cells in future studies. Copyright © 2021 Elsevier Ltd. All rights reserved.

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