Rapid fluorometric assay of LDL receptor activity by DiI-labeled LDL.
DiI-LDL (3,3'-dioctadecylindocarbocyanine-low density lipoprotein) has been extensively used in morphological and microscopic studies of receptor-mediated metabolism of LDL in many cell lines. To date the use of this fluorescent probe in a quantitative assay of LDL receptor activity has not been widely used in studies with multiple samples due to the lack of a practical method for quantitatively recovering cell-associated DiI. Therefore, detection by 125I-labeled LDL has remained the method of choice for assaying LDL receptor activity rapidly and reliably. In this paper, we describe a rapid, simple, and nonradioactive assay of LDL receptor activity using DiI-LDL. The increased sensitivity of this method was achieved by modifications to the labeling procedure of LDL and to the extraction of DiI from cells for subsequent fluorescence determination. These modifications did not affect the affinity of DiI-LDL toward HepG2 cells, and the assay was easily adapted to a rapid screen for LDL receptor modulators in this cell model.
Stephan
Z F
ZF
Research Department, CIBA-GEIGY Corporation, Summit, NJ 07901.
Yurachek
E C
EC
D001667:Binding, Competitive; D002232:Carbocyanines; D006528:Carcinoma, Hepatocellular; D005456:Fluorescent Dyes; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008113:Liver Neoplasms; D008148:Lovastatin / Q000494:pharmacology; D011973:Receptors, LDL / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D005456:Fluorescent Dyes; D006888:Hydroxycholesterols; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; C024286:3,3'-dioctadecylindocarbocyanine; C007997:25-hydroxycholesterol; D008148:Lovastatin