Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay.
Lipoproteins labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) are widely used to visualize LDL-and scavenger-receptor activity in cultured cells. The purpose of this study was to evaluate a new single-step fluorometric assay with high sensitivity for the quantitative determination of the LDL- or scavenger-receptor activity in adherent and non-adherent cells. We used an aqueous solution of 1 g/l SDS dissolved in 0.1 M NaOH to lyse the cells after incubation with DiI-LDL or DiI-acetylated LDL. This allows for the first time the determination of fluorescence intensity and cell protein in the same sample without prior lipid extraction of the fluorochrome. Fluorescence of the cell lysates was determined in microtiter plates with excitation-emission set at 520 and 580 nm, respectively. This rapid method demonstrates high specificity for determining the LDL- and scavenger-receptor activity in cultured cells (e.g., human skin fibroblasts from patients with and without familial hypercholesterolemia; human U-937 monocyte and murine P388 D1 macrophage cell lines). The validity of our fluorescence assay is demonstrated by comparison of cellular uptake and metabolism of lipoproteins labeled with both, DiI and 125iodine. The rapidity and accuracy of this assay allows its routine application for studying receptor-mediated lipoprotein uptake in various cell types.
Institute of Clinical Chemistry, University Hospital Grosshadern, Munich, Germany.
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Teupser
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Thiery
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Walli
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Seidel
D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000378:metabolism; D008264:Macrophages / Q000737:chemistry; D008565:Membrane Proteins; D051379:Mice; D009000:Monocytes / Q000737:chemistry; D011971:Receptors, Immunologic; D011973:Receptors, LDL / Q000032:analysis* / Q000378:metabolism; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B; D012867:Skin; D013050:Spectrometry, Fluorescence
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C024286:3,3'-dioctadecylindocarbocyanine