Measuring Cardiomyocyte ROS Production

A cross-sectional area of each of the isolated single PV cardiomyocytes was imaged using a confocal laser scan microscope (Zeiss LSM 510), and the acquired images were processed using ImageJ measurement tools. We used CellROX Green (Life Technologies, Grand Island, NY, USA) to assess cytosolic ROS production in the freshly isolated control and MIF-treated PV cardiomyocytes at 1 Hz pacing. The measurements were performed using a laser-scanning confocal microscope (Zeiss LSM 510) and an inverted microscope (Axiovert 100) with a 63 × 1.25 numerical aperture oil immersion objective, as described previously.^{7 (link)} Freshly isolated PV cardiomyocytes were maintained in normal Tyrode’s solution (NaCl, 137 mM; KCl, 5.4 mM; CaCl₂, 1.8 mM; MgCl₂, 0.5 mM; HEPES, 10 mM) with 10 µM CellROX Green fluorescent dye. The CellROX Green dye was excited at 488 nm, and fluorescent signals were acquired at wavelengths of >505 nm in the XY mode of the confocal system. The acquired fluorescent images were analysed using Image-Pro Plus 6.0 and Sigma Plot 12 software, as described previously.⁹