Evaluation of a high-content screening fluorescence-based assay analyzing the pharmacological modulation of lipid homeostasis in human macrophages.
BACKGROUND
For understanding cholesterol and phospholipid efflux pathways there is a need for cellular fluorescence-based high-content screens (HCS) to investigated the cholesterol and phospholipid content in human macrophages.


METHODS
Making use of fluorescence imaging based on HCS we have developed a tool to evaluate new agents that can act as inducers of cholesterol efflux. The fluorescence assay is based on the different staining patterns of cholesterol-loaded (E-LDL) and deloaded (HDL3) differentiated monocytes by the saturated, fluorescent lipid probe (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine)-tetramethyl-rhodamin.


RESULTS
Morphologic examination and statistical evaluation of the staining pattern such as gray value, threshold area, shape factor and the spot size distribution provides evidence for a significant pattern change when cholesterol enriched and cholesterol depleted differentiated monocytes were imaged.
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany.
Tobias
T
Werner
Gerhard
G
Liebisch
Margot
M
Grandl
Gerd
G
Schmitz
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D050356:Lipid Metabolism / Q000187:drug effects*; D008075:Lipoproteins, HDL / Q000494:pharmacology; D053440:Lipoproteins, HDL3; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry
D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D008077:Lipoproteins, LDL; D010714:Phosphatidylethanolamines; D012235:Rhodamines; C005358:tetramethylrhodamine; D002784:Cholesterol; C018525:1,2-dimyristoylphosphatidylethanolamine