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  • Title: Adaptation of an in vitro phospholipidosis assay to an automated image analysis system.
    Author: Schurdak ME, Vernetti LA, Abel SJ, Thiffault C.
    Journal: Toxicol Mech Methods; 2007; 17(2):77-86. PubMed ID: 20020975.
    Abstract:
    ABSTRACT Phospholipidosis is the excessive intralysosomal accumulation of phospholipids and is induced in humans and animals by the chronic administration of cationic amphiphilic drugs. To identify compounds that may induce phospholipidosis early in the discovery process, we have developed a predictive fluorescent cell-based assay amenable to automated high content screening using the 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (ss-BODIPY C(12)-HPC) dye and primary rat hepatocytes. ss-BODIPY C(12)-HPC localized to lysosomes that accumulate phospholipids and not to lipid droplets, indicating the selectivity for phospholipid-containing granules. Accumulation of ss-BODIPY C(12)-HPC was monitored in primary rat hepatocytes plated onto 96-well plates and 24 h after exposure to increasing concentrations of 13 drugs known to induce phospholipidosis and four negative compounds. Fluorescent images were captured and analyzed using the Discovery-1 automated cellular imaging system. Eleven out of the 12 selected positive compounds and all negative compounds were properly assigned as positive and negative inducers of phospholipidosis, respectively, indicating the high degree of sensitivity and specificity of this assay. The ability of ss-BODIPY C(12)-HPC to detect and quantify phospholipidosis is similar to that of the well-established probe, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine (NBD-PE).
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