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Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy
E Koren1, J Franzen, R D Fugate
1Oklahoma Medical Research Foundation, Oklahoma City 73104.
Abstract:
Low density lipoprotein (LDL) induced accumulation of cholesterol esters was analyzed by the digital imaging fluorescence microscopy (DIFM) in murine tumor macrophages. To analyze cholesterol ester accumulation, P388D1 macrophages were incubated with increasing quantities of unmodified or acetylated human LDL, washed, and live stained with a lipophylic fluorescent dye Nile Red. The increase in fluorescence intensity was quantitatively determined by the interactive laser cytometer (ACAS 470) and compared with the accumulation of cellular cholesterol esters determined by the gas liquid chromatography. Correlation between the two methods was highly significant (r greater than 0.9, P less than 0.001). A good agreement between the two methods was also found in terms of sensitivity and reproducibility. With the use of 589 nm narrowband interference filter in the light path of emitted light the intensity of fluorescence correlated well with cellular cholesterol ester content even in the presence of relatively high concentrations of triglycerides. Therefore, digital imaging fluorescence microscopy appears to be a reliable method for quantification of cholesterol ester accumulation at the single cell level offering new possibilities of studying interactions between cells and cholesterol ester rich lipoproteins.
Insights
Digital imaging fluorescence microscopy (DIFM) reliably quantifies cholesterol ester accumulation in macrophages. This method offers a sensitive and reproducible alternative to traditional techniques for studying lipoprotein interactions.
Area of Science:
- Cell Biology
- Biochemistry
- Microscopy
Background:
- Low density lipoprotein (LDL) plays a critical role in cellular cholesterol homeostasis.
- Understanding cholesterol ester accumulation in macrophages is vital for studying lipid metabolism and related diseases.
- Traditional methods for quantifying cholesterol esters can be labor-intensive and lack single-cell resolution.
Purpose of the Study:
- To evaluate digital imaging fluorescence microscopy (DIFM) as a method for quantifying cholesterol ester accumulation in macrophages induced by LDL.
- To compare the accuracy, sensitivity, and reproducibility of DIFM with gas-liquid chromatography (GLC).
Main Methods:
- P388D1 murine tumor macrophages were incubated with varying concentrations of unmodified or acetylated human LDL.
- Cells were live-stained with Nile Red, a lipophilic fluorescent dye.
- Fluorescence intensity was measured using an interactive laser cytometer (ACAS 470).
- Results were compared with cholesterol ester content determined by gas-liquid chromatography.
Main Results:
- A highly significant correlation (r > 0.9, P < 0.001) was observed between DIFM and GLC methods.
- DIFM demonstrated good agreement with GLC in terms of sensitivity and reproducibility.
- Fluorescence intensity accurately reflected cholesterol ester content, even with high triglyceride concentrations, using a specific interference filter.
Conclusions:
- Digital imaging fluorescence microscopy (DIFM) is a reliable and sensitive method for quantifying intracellular cholesterol ester accumulation at the single-cell level.
- DIFM provides a valuable tool for studying cellular interactions with lipoproteins, particularly cholesterol ester-rich lipoproteins.
- This technique offers advantages over traditional methods, enabling more detailed investigations into lipid metabolism in macrophages.