The Cell Biology and Imaging Core (Core B) which will be used by all of the Projects will be involved in? three primary functions:? 1) preparation of neonatal rat ventricular myocytes (NRVMs)? 2) preparation of adult mouse ventricular myocytes (AMVMs)? 3) live cell imaging studies? 4) distribution and analysis of results through use of the Open Microscopy Environment (OME) in? coordination with The Scripps Research Institute (TSRI)? The Cell Biology and Imaging Core (Core B) which will be used by all of the Projects will be involved in three? primary functions: 1) preparation of neonatal rat ventricular myocytes (NRVMs), 2) preparation of? adult mouse ventricular myocytes (AMVMs), and 3) live cell imaging studies. Consistent preparation of? cardiomyocytes is critical to the ability to integrate findings across the Projects in the Program. Accordingly,? while many of the individual laboratories have experience or capability in this regard, Core B will serve as a? repository to insure cell availability and cross integration. Preparation of stable neonatal rat ventricular? myocytes is relatively simple, but experimental observations are highly dependent on the methodology in? particular the separation of cardiomyocytes from fibroblasts, plating density and choice of culture medium.? With regard to adult mouse ventricular cardiomyocytes the mechanics and success of the preparation,? and in particular the yield and long term viability of these cells can require skill and consistency. A dedicated? technician and facility is therefore prerequisite to providing cells from TG and KO mice as required by? virtually every project. Core C will also facilitate in the development of methods for expressing genes through? adenoviral infection and expressing proteins through preparation and delivery of TAT fusion proteins. Live? cell imaging will also be performed by Core B. In particular, technical advice and assistance with? experiments using the FRET based Akt activity probe BKAR will be provided, as will analysis of? mitochondrial membrane potential (TMRE) and integrity (RIRR, Calcein). Support for analysis of autophagy? and mitophagy using TAT or adenoviral LC3 or cardiomyocytes from LC3 transgenics will also be provided.? Inverted fluorescence and confocal microscopes equipped for these analyses are available at both UCSD? and TSRI. The Open Microscopy Environment (OME) operated through TSRI provides a unique opportunity? for data sharing and analysis.
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