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One of the major limitations of flow cytometry (FCM) is the absence of an intracellular view. Automated microscopy and image analysis, together with technological developments, led to new approaches in cytometry that bypass the above limitation, introducing high resolution, high content, and large statistical sampling. However, few attempts have been made, until now, to translate the wide repertoire of FCM assays into high-content image screening. This unit describes the implementation of an acquisition and analysis protocol for evaluation of the cell cycle by automated microscopy. The approach grants the possibility to perform simultaneous analysis of a high number of different parameters. A large part of this unit is devoted to the description of hardware features that can optimize the recorded information together with the acquisition and analysis procedures employed to produce good-quality data. Curr. Protoc. Cytom. 70:7.41.1-7.41.15. © 2014 by John Wiley & Sons, Inc. Keywords: image cytometry; cell cycle; S phase; high-content analysis; high resolution; automated microscopy; wide-field microscopy

Optical fluorescence microscopy offers a wide range of technological solutions to address many questions in biomedical research. Spatial resolution has been greatly improved by the use of confocal microscopes, providing a 3-D analysis of the intracellular space. Automation has contributed to make confocal analysis available for high-content image cytometry studies. However, the storage, browsing, and analysis of the amount of data generated can challenge the feasibility of such studies. Presented in this chapter is a multistep acquisition and analysis protocol that can bypass such difficulties by an analysis-driven data collection. Cell-cycle analysis of low-resolution data can be employed to select cell populations of interest that can then be imaged at extremely high resolution and subjected to high-content analysis.

Histone acetylation affects chromatin structural organization, thus regulating gene expression and DNA-related cellular events. Levels of histone acetylation are tightly modulated in normal cells and frequently altered in tumors. Consequently, histone deacetylase inhibitors are currently being tested in clinical trials as anticancer drugs. Presented here is a protocol for measuring the degree of cellular histone tail acetylation, alone or in combination with DNA content to simultaneously evaluate cell ploidy and/or cell cycle progression. The procedure can also be employed to stain peripheral blood samples in order to assess mean histone acetylation levels in patients treated with histone deacetylase inhibitors. Curr. Protocol. Cytom. 46:7.35.1-7.35.8. © 2008 by John Wiley & Sons, Inc. Keywords: flow cytometry; histone; acetylation; histone deacetylase inhibitors; cell cycle; leukemia; clinical trials