The Core provides the necessary expertise and state-of-the-art equipment for rigorous and comprehensive experimental evaluation of cell and tissue specimens to investigate the pathogenic mechanisms of liver disease. The centralized state-of-the-art equipment includes Cellomics ArrayScan® VTI HCS Reader for automated microscope based high content screening and the Seahorse XF96 extracellular flux analyzer for cellular bioenergetics and metabolism studies in real time.
Aims of the Core
- To foster the development and success of proposed COBRE projects and future pilots. The Core will provide appropriate services to the COBRE projects to ensure the validity and relevance of the research findings to advance the understanding of pathogenic mechanisms of liver disease that would be essential for the support and development of individual R01 projects.
- To play an active and significant role in the education and training of the investigators. The Core is committed to the continued education of its users and the University research community at large. The Core hosts several opportunities (e.g., journal clubs, seminars, etc) that serve as forums for scientific and technical interactions between Center users and the UofL research community at large. The Core also disseminates technical and instructional information across the University’s biomedical community to enhance the potential utility of this Core and supported research.
- To assist and participate in the development of new experimental protocols and techniques. The Core’s scope is not limited to the current Projects and studies. With the incorporation of new investigators into the COBRE in the future, the Core will make continual efforts to upgrade its technical abilities to remain on the cutting edge.
- To be an integral and self-sustaining component of the COBRE. The Core is structured to an independent, self-supporting entity. To this end, a fee-for-service plan is implemented for users to support their research.
Facilities & Instrumentation
The Core is housed in a 2500 ft² laboratory space in the CTR Building at the Health Science Center at the University of Louisville and is part of the 20000 ft² of Hepatobiology and Toxicology COBRE contiguous laboratories (open lab concept). Adequate power, gas, vacuum, water and safety equipment (e.g., fume hoods) are available in both locations to carry out procedures and accommodate personnel and projects supported by this Core.
- Seahorse XF96 extracellular flux analyzer
- This platform allows determination basal oxygen consumption, glycolysis rates, ATP turnover and respiratory capacity in a single experiment to assess mitochondrial dysfunction. The XF Analyzer also measures fatty acid oxidation and metabolism of glucose and amino acids for kinetic metabolic information. The Seahorse has the capacity to inject up to 4 different test substances to each well over the course of an experiment, allowing detailed characterization of energy metabolism by mitochondrial and extramitochondrial pathways.
- Thermo Scientific ArrayScan VTI HCS (Cellomics)
- Incorporates the resolving power of microscopic image-analysis on a microtiter plate assay format to allow for much higher throughput that is fully automated.
- Millipore Milliplex analyzer for multianalyte fluorescence determination
- Using flow cytometry-based bead isolation, two lasers and high-speed digital-signal processors, the analyzer reads signals on each individual microsphere particle. It is a particularly powerful platform for simultaneous, ‘multiplex’ detection of proteins in a single biological sample.
- BD FACS Aria III cell sorter/flow cytometer
- The BD FACS Aria III is a powerful instrument designed to separate and collect unique subpopulations of cells from complex mixtures of cells. The instrument is capable of high throughput experimentation and can interrogate up to 90000 cells per second for desired bioactivity.
- BD FACSCanto II flow cytometer
- The system brings BD flow cytometry and multicolor analysis to a new level to help researchers meet complex challenges and advance their research. The BD FACSCanto II provides the ultimate in flexibility and can be configured with two or three lasers to detect up to eight colors. For analysis, the software includes powerful features such as hierarchical snap-to gating, a variety of plots, and batch analysis.
- Applied Biosystems 7900 HT Fast Real Time and 7500 real time PCR systems
- The 7900HT System PCR system detects and quantitates nucleic acid sequences. User-interchangeable Fast 96-well and 384-well TaqMan® Low Density Array blocks are currently available. Each TaqMan Low Density Array “card” available for purchase to use with the latter block includes 384 amplification wells that are pre-loaded with TaqMan® Gene Expression assays. Each Array “card” may be used to evaluate from one to eight cDNA samples.
- Waters 515 HPLC pump with waters 2475 multi λ fluorescence detector
- The 515 HPLC pump is a programmable for flow rate calibration, allows uniform eluent delivery, with highly reproducible peak performance and easy to read display settings.
- Waters 717 plus autosampler with waters 2487 dual λ absorbance detector
- The Waters 717 Plus Autosampler injects samples into a an HPLC systems and automates sampling by storing up to 96 sampling methods or steps. The Waters 2487 Dual λ Absorbance Detector is a two-channel, tunable, ultraviolet/visible (UV/Vis) detector which increases the optical throughput and sensitivity, and reduces bandwidth.
- ChemiDoc XRS+
- The ChemiDoc XRS+ system enables direct digital visualization of chemiluminescent, fluorescent and colorimetric signals. It provides accurate images of accumulated signal and reliable quantitative data for chemiluminescent western blots, as well as enabling image analysis and documentation of protein and DNA/RNA gels and other sample types.
- BioTek Synergy HT Multi-Mode Plate Reader
- This plate reader reads fluorescence, absorbance and luminescence measurements. The reader can read any plate format up to 384-wells with Temperature control up to 50°C.
- Applied Biosystems SOLiD™ 4 Sequencing System
- Enables massively parallel sequencing of DNA fragments which allows for rapid whole genome sequence analysis, and unbiased quantitative determinations of mRNA abundance. The 5500xl has two flowchips, each with six independently addressable and configurable lanes, allowing genome, exome, transcriptome, as well as other sequencing experiments in parallel within the same run.
Additionally, the Core houses centrifuges, computers, refrigerators and freezers, chemical hoods as well as tissue culture hood for sterile techniques, incubators, and all the essential general-use equipment.
The Core will work with investigators to design and perform assays for individual needs.