Measuring secretory membrane traffic: a quantitative fluorescence microscopy approach.

In this chapter the authors describe automated imaging methods to quantify the transport rates of transmembrane as well as soluble cargo, and to evaluate the integrity of the Golgi complex. The quantification of cargo transport rates serves as an example of fluorescence intensity-based assays, the quantification of the Golgi complex integrity--as an example of morphology-based assays. These quantitative assays could be applied for single experiments as well as for middle- and high-throughput screening approaches. Each of these assays can be used to appreciate effects caused by gene silencing by RNAi, cDNA overexpression or application of chemical compounds. For each assay the authors discuss protocols for sample preparation, parameters for automated image acquisition, strategies of image analysis, and data quantification.