Luciferase reporter constructs

Luciferase reporter constructs
Welcome to a Medical Battery specialist of the Nihon Kohden Battery
The protocol employed for cardiomyocyte isolation and culture is a modified version of that described by Radisic et al. (9,10). On day 1, hearts were obtained from 0–3 day old Lewis rats locally bred at the animal facility of Maastricht University. The use of animals for this study was evaluated and approved by the institute's animal ethical committee. On day 2, cardiomyocytes were pre-plated for 2 h an subsequently plated at >30,000 cells/cm2 on plastic (Permanox, Lab-Tek; Nunc, Langenselbold, Germany) chamber slides and incubated overnight in high-serum medium supplemented with 10 µmol/L cytosine arabinoside (Ara-C; Cat. no. C1768; Sigma-Aldrich, St. Louis, MO, USA) to further decrease the number of actively dividing (mostly non-myocyte) cells (11,12).
The Permanox chamber slides did not require coating since cardiomyocytes readily adhere to them with battery such as Nihon Kohden BSM-2353C Battery, Nihon Kohden BSM-2301K Battery, Nihon Kohden BSM-2351C Battery, Nihon Kohden BSM-2303C Battery, Alaris Medicalsystems SIGNATURE 2 Battery, Alaris Medicalsystems 7000 Battery, Alaris Medicalsystems 7100 Battery, Alaris Medicalsystems 7130 Battery, Alaris Medicalsystems 7200 Battery, JMS 7N-1200SCK Battery. The culturing parameters were empirically determined through pilot experiments which revealed that the use of glass-based chamber slides with surface coatings like Histogrip (Invitrogen, Carlsbad, CA, USA) and especially Matrigel (BD Biosciences, San Jose, CA, USA) led to dramatic changes in cardiomyocyte cellular structure and function; cardiomyocytes remained rounded, resulting in diminished intercellular connections and fewer contractions (data not shown). At densities 30,000 cells/cm2 without electrical stimulation, the cells displayed no contractions and remained round. Over 50% of these cells died within 2 days of culture, most likely because they were not able to form the connections required for intercellular signaling.
The resulting cultures consisted of >95% cardiomyocytes (purity of cultures can be observed in Figure 2). On day 3, the medium was replaced with Ara-C–free high-serum medium since Ara-C was shown to directly interfere with the contractile activity of the cells (data not shown). For transfection studies, cells can be transfected at this time. We obtained reproducible results for luciferase reporter constructs with the transfection reagent Fugene HD (Roche) using the manufacturer's protocols when transfecting cells in high-serum medium. In our studies we employed transfection studies to examine the promoter sequences of cardiac genes. A Myh6 promoter–luciferase reporter construct was transfected into cardiomyocytes which subsequently were electro-stimulated (Figure 2). The behavior of the luciferase reporter construct is similar to that of the endogenous Myh6; the activation observed in Figure 2 is believed to be electrical pulse–dependent.