Combination Of Fluorescent Labeling Research Articles

On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates.

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.

Postnatal Migration of Cerebellar Interneurons.

Due to its continuing development after birth, the cerebellum represents a unique model for studying the postnatal orchestration of interneuron migration. The combination of fluorescent labeling and ex/in vivo imaging revealed a cellular highway network within cerebellar cortical layers (the external granular layer, the molecular layer, the Purkinje cell layer, and the internal granular layer). During the first two postnatal weeks, saltatory movements, transient stop phases, cell-cell interaction/contact, and degradation of the extracellular matrix mark out the route of cerebellar interneurons, notably granule cells and basket/stellate cells, to their final location. In addition, cortical-layer specific regulatory factors such as neuropeptides (pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin) or proteins (tissue-type plasminogen activator (tPA), insulin growth factor-1 (IGF-1)) have been shown to inhibit or stimulate the migratory process of interneurons. These factors show further complexity because somatostatin, PACAP, or tPA have opposite or no effect on interneuron migration depending on which layer or cell type they act upon. External factors originating from environmental conditions (light stimuli, pollutants), nutrients or drug of abuse (alcohol) also alter normal cell migration, leading to cerebellar disorders.

Tu1878 Establishment of a Severely Cachexic Rat Model With Possible Ghrelin Resistance by Using a Novel 85As2 Cell Line Developed by Repeated Peritoneal Dissemination After Orthotopic Implantation of the Human Gastric Cancer Cell Line Mkn-45

G A A b st ra ct s time, neural innervation appeared to be disrupted in early PanIN lesions. Conclusions: UEAI is a specific cell surface marker to bind the PanIN. Alterations in vascularization and innervation are prominent in PanIN bearing-mice and may contribute to the earliest events in PanIN formation. The combination of fluorescent labeling and 3-D microscopy provides a new method to visualize PanIN to aid early detection and diagnosis of pancreatic lesions and cancer. Further research on the serial evolution of these changes may provide insight into the pathogenesis of pancreatic cancer.

Cell Coat Mediated Cell Migration

Cell migration depends on a sequence of adhesion and detachment events. These events arise during the cyclic migration process, which involves the integrin-dependent adhesion machinery, the acto-myosin system and the signaling pathways between them. Migration appears to rely on a delicate spatio-temporal regulation of cell adhesion to the surrounding substrate. Studies of cell migration in physiological contexts have shown that a pericellular coat, a thickened polymer matrix attached to many cell surfaces, often is required to facilitate cell migration, including that of aggressively spreading cancers. Cell motility in these systems directly depends on the formation of a large, hyaluronan-rich cell coat with an asymmetric distribution around the polarized migrating cell. Removal or alteration of the coat substantially decreases motility - to the point that such treatments have been proposed as therapies for some types of cancer. The hyaluronan biology community often speculates that cell migration requires the ‘insulation’ and/or the mechanical properties provided by the cell coat. However, little has been done to substantiate this claim. A bigger problem yet is that the lubricating effect of hyaluronan has been shown to oppose adhesion, which leads to a conundrum in the present context: How is it possible that inhibition of adhesion can help a cell migrate, when adhesion is absolutely necessary to gain traction and exert the force to move the cell forward? We have developed a microfluidics-based cell migration assay capable of presenting several surface gradients of fibronectin of different slopes to induce cell migration within the same device. We study the ability of these gradients to induce cell migration and their influence on the cell coat phenotype and mechanical properties using a combination of fluorescent labeling, particle exclusion assays, and optical tweezer force probe experiments.