Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy.
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.
Koren
E
E
Oklahoma Medical Research Foundation, Oklahoma City 73104.
Franzen
J
J
Fugate
R D
RD
Alaupovic
P
P
D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000097:blood; D002788:Cholesterol Esters / Q000378:metabolism*; D002849:Chromatography, Gas; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007941:Leukemia P388 / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D010078:Oxazines; D012815:Signal Processing, Computer-Assisted; D013050:Spectrometry, Fluorescence; D014280:Triglycerides / Q000097:blood
D002788:Cholesterol Esters; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D010078:Oxazines; D014280:Triglycerides; D002784:Cholesterol; C044808:nile red