title: Live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy.
abstract: Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope's autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.
authors.affiliation: Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology; Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine; takashi.sakurai@mpi-muenster.mpg.de.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
authors.forename: Anthony
authors.initials: A
authors.lastname: Lanahan
authors.affiliation: Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Woolls
authors.affiliation: Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
authors.forename: Daniela
authors.initials: D
authors.lastname: Tirziu
authors.affiliation: Department of Internal Medicine, Yale Cardiovascular Research Center and Section of Cardiovascular Medicine.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Murakami
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D007091:Image Processing, Computer-Assisted / Q000379:methods; D016086:Lentivirus / Q000235:genetics*; D018613:Microscopy, Confocal / Q000379:methods*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D051381:Rats; D014161:Transduction, Genetic / Q000379:methods*