No abstract

Long term monitoring of live cells in vitro or in vivo presents considerable challenges. Fluorescent tracers are used as one of the most common methods. However, fluorescent molecules usually are limited by cytotoxicity and/or lack of persistence within live cells over sufficient time periods. In this study, we evaluated our newly developed cell tracing/tracking reagents, CellTrac Far Red and CellTracker Deep Red, as novel red laser-excitable fluorescent dyes for flow cytometry and live cell imaging applications. These dyes cross the plasma membrane and covalently bind proteins inside cells, where the stable, well-retained fluorescent label offers a consistent, reliable fluorescent signal without affecting morphology or physiology and also exhibiting low cellular toxicity. CellTrace Far Red was demonstrated to be an excellent probe for cellular proliferation studies using flow cytometry, allowing measurement of up to eight generations of proliferating cells. CellTracker Deep Red was shown to be an excellent reagent for the study of cell movement, location and morphology by labeling and imaging live-cells with fluorescence microscopy. In addition, CellTracker Deep Red has excellent cell retention characteristics with bright fluorescence intensity even after 72 hours of cell staining, with signal retained after fixation, and can be multiplexed with other fluorescent dyes.

Automated quantitative fluorescence microscopy, also known as high content imaging (HCI), is a rapidly growing analytical approach in cell biology. Because automated image analysis relies heavily on robust demarcation of cells and subcellular regions, reliable methods for labeling cells is a critical component of the HCI workflow. Labeling of cells for image segmentation is typically performed with fluorescent probes that bind DNA for nuclear-based cell demarcation or with those which react with proteins for image analysis based on whole cell staining. These reagents, along with instrument and software settings, play an important role in the successful segmentation of cells in a population for automated and quantitative image analysis. In this chapter, we describe standard procedures for labeling and image segmentation in both live and fixed cell samples. The chapter will also provide troubleshooting guidelines for some of the common problems associated with these aspects of HCI.

High content screening (HCS)-based multiparametric measurements are very useful in early toxicity testing and safety assessment during drug development, and useful in evaluating the impact from new food supplements and environmental toxicants. Mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phosphoplipidosis, and steatosis are a few of the important markers routinely studied for the assessment of drug-induced liver injury and toxicity. Mitochondrial dysfunction leads to oxidative stress and cell death. Liver injury from drug-induced phospholipidosis and steatosis is routinely studied in hepatotoxicity investigations to determine the risk factors and fate of drugs or chemical compounds as some drugs can lead to defects in lipid metabolism and accumulation of lipids in lysosomes. In this chapter, we describe fluorescent reagents and the protocols for the measurement of various parameters such as mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phospholipidosis, and steatosis using high content imaging-based methodologies and instrumentation.