Cell & Molecular Imaging Core
John J. Lemasters, M.D., Ph.D.
Professor of Drug Discovery & Biomedical Sciences
Professor of Biochemistry & Molecular Biology
GlaxoSmithKline Distinguished Endowed Chair in Advanced Cellular Technologies
The Cell and Molecular Imaging Core provides major support in confocal, multiphoton, and super-resolution microscopy to COBRE investigators. The Core houses five major microscope systems:
- Zeiss LSM 880 NLO multiphoton/confocal system with Airyscan super-resolution capability equipped with a Coherent Chameleon tunable femtosecond Ti-Sapphire laser and Quasar spectral detection
- BD Biosciences CARV II real-time spinning disk confocal microscope equipped with a Photometrics Cascade 1K Digital camera and IPLab image processing and analysis software
- Zeiss LSM 510 laser scanning confocal microscope with META spectral detection
- Olympus Fluoview FV1200 intravital multiphoton microscope equipped with a Spectra-Physics Mai Tail tunable Ti-Sapphire laser
- Olympus Fluoview FV10i laser scanning desktop microscope.
The Zeiss and the Olympus microscope systems are equipped with environmental chambers for temperature and gas phase control to allow high resolution non-destructive 3-dimensional imaging of living cells, tissues, small animal organs, and organisms. The Olympus Fv10i system has hypoxia control as well.
Lauren Ball, Ph.D.
Cell and Molecular Pharmacology and Experimental Therapeutics
The Mass Spectrometry Facility and REDOX Proteomics Core within the Medical University of South Carolina Proteomics Center provides expertise, services, education, and training to enhance biomedical research endeavors through the mass spectrometry-based proteomics. Protein analysis includes proteolytic digestion, chromatographic separation and tandem mass spectrometric analysis (LC-MS/MS), and interpretation of tandem mass spectra using protein database-searching algorithms. The facility assists in the development of customized applications for affinity chromatography and characterization of post-translationally modified peptides (e.g. S/T/Y phosphorylation, S/T O-GlcNAc modification, Cys oxidation and S-glutathionylation, N- and O-linked glycosylation, Lys acetylation, ubiquitylation, and non-enzymatic glycation). Quantitative proteomic approaches using stable isotope labeling (SILAC), label free proteomics (MaxLFQ, Skyline), or isobaric tagging (iTRAQ, ICAT, TMT) are available to identify differentially expressed proteins and/or differentially regulated sites of post-translational modification from cell lines or tissue samples.
Bioenergetics Profiling Core
Craig Beeson, Ph.D.
Associate Professor of Pharmaceutical and Biomedical Sciences
The primary goal of the Bioenergetics Profiling core facility is to support COBRE investigators in the characterization of metabolite fluxes related to cellular redox and primary energy metabolism – the prevalent sources of both the primary and secondary cellular redox species. The facility provides access to traditional, ‘gold standard’ techniques such as isotopomer, radiometric, and spectroscopic analyses. The core is also a development site for the Seahorse Biosciences extracellular flux (XF) fluorometric technology used to measure metabolic fluxes (i.e., oxygen consumption, CO2, and lactate extrusion) in real time using multiwell plates. The basic Seahorse XF applications enable high throughput metabolic measurements with small sample sizes that have transformed the utility of quantitative analyses of metabolic fluxes. Innovative adaptations of the XF technologies developed in the core facility are providing access to real time flux measurements of redox species in cells and tissues and, more importantly, the interrogation of bioenergetics pathways via use of pharmacological or genetic interventions. We have coordinated these recent strategic technological acquisitions into a core that provides analytical support for the efforts of the COBRE investigators and their collaborators while also extending the technology to suit new efforts and further enable measurements with improved translational potential.
Analytical Redox Biology Core
Danyelle Townsend, Ph.D.
Drug Discovery and Biomedical Sciences
Understanding the complexities of redox mediated signaling events requires a multidisciplinary approach. Analytical biochemistry specific to the detection and quantification of redox sensitive molecules and coordinate protein changes that drive homeostasis is a unique niche. The primary objective of the Analytical Redox Biology Core (ARBC) is to provide comprehensive analytical redox biochemistry methods and mentoring support for the COBRE junior faculty with the goal to advance their research endeavors, publications, and fundability. The specific aims of the ARB Core are:
- Provide ROS /RNS identification and quantification using state-of-the-art techniques
- Perform quantitative analysis of ROS/RNS (redox molecules and metabolites), including those associated with calcium mobilization and changes in energy metabolism
- Provide expertise and technology for in depth biochemical analysis of thiol-centered enzyme activities and define protein:protein interactions.
Since oxidative (nitrosative) stress often is associated with a conditional increase in antioxidant protection, the Core has established methods to detect and measure various antioxidant enzyme activities as a function of oxidant stress/antioxidant protection equilibrium. Comprehensive analysis of redox status also includes measurement of intracellular GSH, GSSG, protein surface and “buried” thiols utilizing both endpoint and/ or real-time kinetic measurements with millisecond resolution. In complex studies of redox signaling, certain protein- protein interactions appear to be redox dependent and attributed to post-translational modifications, including S-nitrosylation and S-glutathionylation.