Inverted fluorescence microscope

H9C2 cells were fixed with 4% paraformaldehyde for 15 min and were stained with Hoechst 33258 (Biyuntian Biotechnology Co., Ltd., China) for 30 min in the dark. Next, H9C2 cells were observed under an inverted fluorescence microscope (Leica Microsystems GmbH).
The ROS assay was conducted according to the manufacturer’s instructions of the ROS Assay Kit bought from Biyuntian Biotechnology Co., Ltd. Next, 10 μM DCFH diluted in DMEM was added to each well, followed by incubation for 30 min at 37°C. The cells were then observed under an inverted fluorescence microscope (Leica Microsystems GmbH).

[Ca²⁺]_cyt imaging experiments were performed as previously described (52 ). Briefly, cells were grown on glass coverslips for 24 h and incubated with 5 μM fura-2/AM (Invitrogen) for 1 h in physiological salt solution (PSS) at 37 °C humidified atmosphere containing 5% CO₂ in the dark and then washed with PSS for 20 min. Then, cells on coverslips were mounted in a standard perfusion chamber on the stage of an inverted fluorescence microscope (Leica). Fluorescence signals were imaged using an intensified CCD camera (ICCD200) attached to an inverted fluorescence microscope (Leica) and recorded with MetaFluor software (Universal Imaging Corporation). Images were acquired every 3 s. The dual wavelength excitation method for the measurement of fura-2 fluorescence was used. The excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was collected at 510 nm. [Ca²⁺]_cyt was presented as fluorescence ratios (F340/F380) after background subtraction. The PSS contained the following: 140 mM Na⁺, 5 mM K⁺, 2 mM Ca²⁺, 147 mM Cl⁻, 10 mM Hepes and 10 mM glucose (pH 7.4). The 0 Ca²⁺ solution (0 Ca²⁺) contained the following: 140 mM Na⁺, 5 mM K⁺, 145 mM Cl⁻, 0.5 mM EGTA, 10 mM Hepes, and 10 mM glucose (pH 7.4). The osmolality for the solution was ∼300 mosmol^.kg⁻¹ of H₂O.