Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here, we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the <i>Gaussia</i> luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The <i>Gaussia</i> luciferase mycosensor assay provides an easy tool to monitor mammalian cell contaminants in a high-throughput fashion

Multiplex blood reporters for simultaneous monitoring of cellular processes

Contains fulltext : 125685.pdf (publisher's version ) (Open Access)Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted Vargula luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted Gaussia luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously

Sensitive Assay for Mycoplasma Detection in Mammalian Cell Culture

Mycoplasma contamination in mammalian cell cultures is often overlooked yet is a serious issue which can induce a myriad of cellular changes leading to false interpretation of experimental results. Here we present a simple and sensitive assay to monitor mycoplasma contamination (mycosensor) based on degradation of the Gaussia luciferase reporter in the conditioned medium of cells. This assay proved to be more sensitive as compared to a commercially-available bioluminescent assay in detecting mycoplasma contamination in seven different cell lines. The Gaussia luciferase mycosensor assay provides an easy tool to monitor mammalian cells contaminants in a high-throughput fashion

Directed Molecular Evolution Reveals Gaussia Luciferase Variants with Enhanced Light Output Stability

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10–15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics

Multiplex Blood Reporters for Simultaneous Monitoring of Cellular Processes

Reporters secreted into the conditioned medium of cells in culture or into blood in vivo have shown to be useful tools for simple and noninvasive monitoring of biological processes in real-time. Here, we characterize the naturally secreted <i>Vargula</i> luciferase as a secreted blood reporter and show that this reporter can be multiplexed with the secreted <i>Gaussia</i> luciferase and alkaline phosphatase for simultaneous monitoring of three different cellular processes in the same biological system. We applied this system to monitor the response of three different subsets of glioma cells to a clinically relevant chemotherapeutic agent in the same well in culture or animal in vivo. This system could be extended to any field to detect multiple processes in the same biological system and is amenable for high-throughput screening to find drugs that affect multiple cellular populations/phenomena simultaneously

Type grouping in rat skeletal muscle after crush injury

Object. Accuracy of reinnervation is an important factor that determines outcome after nerve injury and repair. Type grouping-the clustering of muscle fibers of the same type after reinnervation-can be used to investigate the accuracy of reinnervation. In this study, the degree of type grouping after crush injury in rats was compared with the clustering of muscle fibers after autografting or single-lumen nerve grafting. Methods. Twelve weeks after sciatic nerve crush injury in rats, clustering of Type I muscle fibers was analyzed in the target muscle with adenosine 5'-triphosphatase staining. In addition, the number of regenerated axons was determined in the nerve distal to the crush injury. Results were compared with that of the authors' previous study. Results. Type grouping was more abundant after crush injury than after autograft or single-lumen nerve graft repair. Conclusions. Crush injury leads to more clustered innervation of muscle fibers, probably because the Schwann cell basal lamina tubes are not interrupted as they are in autograft or artificial nerve graft repair. This finding adds to understanding the processes playing a role in nerve regeneration. (DOI: 10.3171/2010.9.JNS091656)Stem cells & developmental biolog

Cell-based Immunotherapy Against Gliomas: From Bench to Bedside

Glioblastoma (GBM) comprises 51% of all gliomas and is the most malignant form of brain tumors with a median survival of 18–21 months. Standard-of-care treatment includes maximal surgical resection of the tumor mass in combination with radiation and chemotherapy. However, as the poor survival rate indicates, these treatments have not been effective in preventing disease progression. Cellular immunotherapy is currently being explored as therapeutic approach to treat malignant brain tumors. In this review, we discuss advances in active, passive, and vaccine-based immunotherapeutic strategies for gliomas both at the bench and in the clinic