High-content screening (HCS) as a methodological tool has evolved relatively recently, largely driven by the demand for in depth spatial and temporal information from intact cells exposed to a range of chemical and/or genomic perturbations. The technology is based around automated fluorescence microscopy in combination with advanced imaging processing and analysis tools, which together can provide quantitative information as a first-level description of complex cellular events. HCS and high-content analysis are particularly powerful when combined with perturbation techniques such as RNA interference (RNAi), as this allows large families of genes to be interrogated with respect to a biological pathway or process of interest. In this methodology chapter, we describe an approach by which HCS can be applied to study the morphological state of the Golgi complex in cultured mammalian cells. We provide a detailed protocol for the highly parallel downregulation of gene activity using RNAi in 384-well plates and describe an automated image analysis routine that could be used to quantify Golgi complex in a genome-wide RNAi context.
Keywords: Golgi structure; High-content analysis; High-content screening; RNAi screen.
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