Cellular and Molecular Physiology Core Facility
This Core facility provides technical expertise, equipment and personnel to Liver Center Investigators who wish to work with: a) Isolated cell preparations including: hepatocytes, cholangiocytes, endothelial cells, stellate cells, portal fibroblasts and hepatic lymphocytes, primarily from rat and mouse. Recently human hepatocytes have also been obtained; b) Cell culture facilities for short and long term cultures and cell lines; c) Isolated liver perfusion preparations for studies utilizing the whole organ; d) Gene expression using Quantitative real time PCR machine and Phosphor-imaging equipment; e) Altering gene expression in these cells and tissues using siRNA transfection and adenovirus infection technologies; and f) access to Animal Models of Disease: expertise is available for the use of rat and mouse animal models of Cholestatic liver disease (bile duct ligation, estrogen administration, LPS administration) and fibrosis and cirrhosis (bile duct ligation, CCL4 and Phenobarbital administration, Schistosomiasis).
By centralizing these procedures in a Core facility, investigators are assured of a high degree of quality control and preparations can often be used simultaneously by more than one investigator.
Cell and Isolation and Organ Perfusion Component
This facility isolates hepatocytes and non-parenchymal liver cells, including bile duct cells, Kupffer cells, Ito cells and endothelial cells from rats and mice, maintains rodent and human hepatocytes in short term collagen sandwich cultures and maintains long-term cultures of cell lines including HepG2, WIF2B, COS-7, and SKHep1 cells. The facility also performs isolated liver perfusions, including normal, cirrhotic and cholestatic livers. A Matrix Anesthesia Machine was recently acquired which has increased the number of animals that can be utilized both for in-vivo studies of bile secretion as well as preparation of isolated liver cell populations. To use this facility, Liver Center investigators should contact Kathy Harry (203-785-3151) for isolated cell preparations or for perfused liver preparations. New investigators should first contact the Core's Director, Dr. Nathanson (203-785-5279).
Molecular Biology Component
This component includes a) Quantitative measurements of mRNA by real time RT-PCR (ABI 7500 real time thermal cycler); b) Fotodyne Imaging Workstation: This instrument uses a CCD camera to photograph gels and blots in either transmitted white and UV light or reflected white light modes. Interested individuals should contact Dr. Boyer (203-785-5279); c) BioTek Synergy 2 Multi-Detection Microplate Reader: This equipment provides fluorescence intensity, time resolved fluorescence, fluorescence polarization, glow and flash luminescence, UV-Vis absorbance, FRET, TR-FRET, BRET, area scanning, and spectral scanning; d) Interfering RNA (siRNA transfections) and 3.4.4 Adenovirus infection; e) Generation of pooled RNA and protein samples from well characterized experimental models and controls; f) QiaCube: this equipment provides for automated processing of plasmid DNA mini-preps, PCR product purification and RNA extraction and clean-up, reducing the bench time required for routine assays; g) Roche lightcycler 480 II system – accelerates PCR analysis and enables genotyping; and h) Access to the Keck Biotechnology Resource Laboratory: The Keck Biotechnology Resource Laboratory provides state-of-the-art biotechnology resources throughout the University. Molecular Sequencing and primer construction are common facilities available to all Yale faculty. The Keck facilities also include advanced biostatistical resources, microarray facilities, and a dedicated proteomics core that are cost-shared with this Center.