Nis elements ar 3

Changes in morphology of H9c2 cells were observed and imaged using an inverted epifluorescence microscope Nikon Eclipse TS100 with 10-40x air objectives (Nikon, Japan) equipped with a digital camera 1300Q (VDS Vosskühler, Germany) and the software NIS-Elements AR 3.10 (Laboratory Imaging, Czech Republic). Furthermore, the cellular viability was visualised using nuclei staining with Hoechst 33342 (Molecular Probes, OR, U.S.A.) and propidium iodide (PI; Molecular Probes, OR, U.S.A.), which are well-established and sensitive probes to determine apoptosis and necrosis: Hoechst 33342 is a blue-fluorescent probe (λ_ex = 360 nm, λ_em = 460 nm) staining all nuclei. In apoptotic cells, chromatin condensation occurs and apoptotic cells can thus be identified as those with condensed and more intensely stained chromatin. The red-fluorescent (λ_ex = 560 nm, λ_em = 630 nm) DNA-binding dye, PI, is unable to cross the plasma membrane of living cells, but readily enters necrotic (or late-stage apoptotic) cells and stains their nuclei red. H9c2 cells seeded in 12-well plates at a density of 75,000 cells per well were incubated with compounds under investigation (alone or in combinations) for 24 h. After that, cells were stained with 10 μg/mL Hoechst 33342 and 1 μg/mL PI for 20 min at room temperature.

Mitochondrial activity was assessed in NVCMs using the JC1 probe (5,5′,6,6′-tetrachlor-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Molecular Probes/Invitrogen, Czech Republic). JC1 becomes non-specifically accumulated in cellular cytosol as a green fluorescent monomer (λ_ex = 480 nm; λ_em = 535 nm). In metabolically-active mitochondria with polarized inner membrane, JC1 monomers flock into red fluorescent J-aggregates (λ_ex = 560 nm; λ_em = 630 nm). Cells were seeded on 24-well plates and incubated for 24 h with tested compounds and then loaded with 3 μM JC1 for 15 min at 37 °C after which the medium was replaced with PBS. Photomicrographs were obtained using the epifluorescence inverted microscope Eclipse TS100 (Nikon, Japan) equipped with digital cooled camera (1300Q, VDS Vosskühler, Germany) and software NIS-Elements AR 3.10 (Laboratory Imaging, Czech Republic).