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Blood-Based Assay with Secreted Gaussia Luciferase to Monitor Tumor Metastasis

Only a few techniques are currently available for quantifying systemic metastases in preclinical models. Cancer cell expression of naturally secreted Gaussia luciferase (Gluc) provides a useful circulating biomarker that enables the monitoring of metastatic tumor burden and ...

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The Bioluminescent Imaging of Spontaneously Occurring Tumors in Immunocompetent ODD-Luciferase Bearing Transgenic Mice

The imaging of spontaneously occurring tumors in mice poses many technical and logistical problems. Recently a mouse model was generated in which a chimeric protein consisting of HIF-1α oxygen-dependent degradation domain (ODD) fused to luciferase was ubiquitously expressed in all ...

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Real-Time Bioluminescent Tracking of Cellular Population Dynamics

Cellular population dynamics are routinely monitored across many diverse fields for a variety of purposes. In general, these dynamics are assayed either through the direct counting of cellular aliquots followed by extrapolation to the total population size, or through the monitori ...

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Monitoring of Transcriptional Dynamics of HIF and NFB Activities

Genetic experiments over the last few decades have identified many regulatory proteins critical for DNA transcription. The dynamics of their transcriptional activities shape the differential expression of the genes they control. Here we describe a simple method, based on the secre ...

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Bioluminescence Reporter Gene-Based Detection of MicroRNAs

MicroRNAs (miRNAs) are an abundant class of small noncoding RNA molecules that inhibit the expression of cognate genes in multicellular organisms. These small RNAs have been demonstrated to play crucial roles in a variety of biological processes including cell differentiation, pro ...

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Video-Rate Bioluminescence Imaging of Protein Secretion from a Living Cell

We have established a method of video-rate bioluminescence imaging to visualize exocytotic protein secretion from a single living cell using Gaussia luciferase (GLase) as a reporter protein. The luminescence signals produced by the enzymatic reaction of secreted GLase (lucifer ...

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Split Gaussia Luciferase for Imaging LigandReceptor Binding

Ligand binding to cell surface receptors activates signaling pathways in normal and pathologic conditions, and internalized ligand–receptor complexes may continue to signal from endosomes. Accessibility of cell surface receptors and the central function of ligand–recept ...

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Fabrication of Bioluminescent Capsules and Live-Cell Imaging

The plasma membrane of living cells is an interface of material transfers and an antenna for outer signals. This chapter provides a guide on how to fabricate bioluminescent capsules for illuminating intracellular signaling and cargo protein delivery. The capsule consists of four compo ...

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Gaussia Luciferase-Based Mycoplasma Detection Assay in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell culture is a common problem with serious consequences on experimental data, and yet many laboratories fail to perform regular testing. In this chapter, we describe a simple and sensitive mycoplasma detection assay based on the bioluminesc ...

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Neuronal Network Imaging in Acute Slices Using Ca2+ Sensitive Bioluminescent Reporter

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca2+ indicators is widely used to record transient intracellular Ca2+ increases associated with bio ...

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Extraction and Quantification of Adenosine Triphosphate in Mammalian Tissues and Cells

Adenosine 5′-triphosphate (ATP) is the “energy currency” of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extrac ...

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Bioluminescence Imaging: Basics and Practical Limitations

Over the last three decades, imaging has been a thriving field with continuous egression of more reliable and highly sophisticated tools and techniques allowing better understanding of biological processes in living organisms. This field continues to expand and its applications br ...

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High-Resolution In Vivo Imaging of Fluorescent Proteins Using Window Chamber Models

Fluorescent proteins enable in vivo characterization of a wide and growing array of morphological and functional biomarkers. To fully capitalize on the spatial and temporal information afforded by these reporter proteins, a method for imaging these proteins at high resolution long ...

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The Use of Fluorescent Proteins for Intravital Imaging of Cancer Cell Invasion

The analysis of cancer cell behavior in the primary tumor in living animals provides an opportunity to explore the process of invasion and intravasation in the complex microenvironment that is present in vivo. In this chapter, we describe the methods that we have developed for performing intr ...

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Assessing Cancer Cell Migration and Metastatic Growth In Vivo in the Chick Embryo Using Fluorescence Intravital Imaging

Cell migration and metastasis are key features of aggressive tumors. These processes can be difficult to study, as they often occur deep within the body of a cancer patient or an experimental animal. In vitro assays are able to model some aspects of these processes, and a number of assays have been develo ...

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Subcellular Imaging In Vivo: The Next GFP Revolution

The use of fluorescent proteins to differentially label cancer cells in the nucleus and cytoplasm and high-powered imaging technology have been used to visualize the nuclear–cytoplasmic dynamics of cancer-cell in vivo. Nuclear–cytoplasmic dynamics have been imaged in cancer cel ...

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In Vivo Imaging of Oligonucleotide Delivery

RNA interference (RNAi) has rapidly become a powerful tool for drug-target discovery and therapeutics. Cancer is an important application for RNAi therapeutics, since abnormal gene regulation is thought to contribute to the pathogenesis and maintenance of the metastatic phenoty ...

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What Does It Take to Improve Existing Fluorescent Proteins for In Vivo Imaging Applications

Although fluorescent proteins are ubiquitously used as genetic tracers and imaging agents, there is significant room for improvement. This chapter discusses how new improved fluorescent proteins can be designed. It focuses on the design of far-red and infrared fluorescent protein ...

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Screening Reef Corals for Novel GFP-Type Fluorescent Proteins by Confocal Imaging

The discovery of multicolored fluorescent proteins (FPs), in reef corals, that are close relatives of the green fluorescent protein (GFP) has led to what is now viewed as the second GFP revolution. Numerous GFP-type proteins, termed “reef FPs,” have been cloned from reef organisms and many posse ...

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In Vivo Imaging of the Developing Mouse Embryonic Vasculature

Live confocal microscopy of vital fluorescent markers, expressed in mouse embryonic tissues, is a powerful and exciting method to study mammalian embryonic development. This chapter discusses imaging approaches to visualize and characterize dynamic changes of the yolk-sac vas ...

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