Millicells ez slide 8 well glass

To determine the effect of EA-Aa on ROS formation in cardiomyoblasts, H9c2 cells (3 × 10⁴ cells.well⁻¹) were seeded in MilliCells^® EZ Slide 8-well glass (Millipore, United States). After reaching 80% of confluence, cells were treated with EA-Aa (125, 250, and 500 μg mL⁻¹) for 30 min, followed by the addition of Dox (IC₂₀ = 0.5 μg mL⁻¹ and IC₅₀ = 1 μg mL⁻¹) overnight. The evaluation of intracellular ROS was carried out with 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA, Invitrogen, United States), following the manufacturer’s instructions. DAPI was used to stain the cell nucleus. Images were obtained with a fluorescence microscope (Zeiss Axio Observer Z1) with an incorporated camera (Zeiss, Germany), detected with 504 nm of excitation and 525 nm of emission for DCF and 353 nm of excitation, and 465 nm of emission for DAPI (Sigma, United States). The settings were the same for all analyses. The quantification was performed in the entire image with the software ImageJ.

In order to assess the role of the EA-Aa in the reduction of ROS formation, 8 × 10⁴ Cos-7 cells were seeded in MilliCells® EZ Slide 8-well glass (Millipore, MA). After 24 h, cells were treated with EA-Aa and H₂O₂ in the same conditions used in the previous antioxidant assay. The evaluation of intracellular ROS was realized with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and with dihydroethidium (DHE) probes, following the manufacturer's instructions (n = 3). DAPI was used to stain cell nucleus. Images were obtained with a fluorescence microscope (Zeiss Axio Observer Z1) with an incorporated camera (Zeiss, Germany), detected with 504 nm of excitation and 525 nm of emission for DCF, 587 nm of excitation and 610 nm of emission for DHE, and 353 nm of excitation and 465 nm of emission for DAPI. The settings were kept constant for all analysis. The entire image was used for quantification, and the analysis was performed with ImageJ software.