2.3. Apoptosis Assay

To detect the apoptosis induced by doxorubicin in H9c2 cells, a FITC Annexin V Apoptosis Detection Kit (Invitrogen-Life Technologies, Carlsbad, CA, USA) was used, and a flow cytometric analysis was performed. Briefly, cells were cultured in 6-well plates at 3 × 10⁵ cells/well and were then treated with doxorubicin (10⁻⁵ M) alone for 6 h, treated with lipid emulsion (0.25%) alone for 7 h, or pretreated with lipid emulsion (0.25%) for 1 h followed by doxorubicin (10⁻⁵ M) for 6 h. Following the treatment, the cells were washed three times with cold PBS (pH 7.4), resuspended in 1× binding buffer and stained with Annexin V-FITC and propidium iodide (PI) for 10 min at room temperature in the dark. Finally, the cells were resuspended in 400 μL of 1× binding buffer and immediately analyzed by flow cytometry. The cell viability and apoptosis rates were measured on a FC-500 flow cytometer (Beckman Coulter, Pasadena, CA, USA) using 488-nm laser excitation and fluorescence emission at 530 nm (FL1) and >575 nm (FL3). A total of 20,000 cells per treatment condition were acquired from three independent experiments and analyzed using Beckman Coulter CPX software (Beckman Coulter, CXP 2.2, Mervue Business Park, Mervue, Galway, Ireland). For forward and side scatter measurements, linear amplification was applied, and logarithmic amplification was used for all fluorescence measurements. Quadrant analysis was performed on the gated fluorescence dot plot to quantify the percentages of live, necrotic and apoptotic cell populations.

To detect whether doxorubicin induces late apoptosis in H9c2 cells, a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay was conducted as previously reported [16,17]. Briefly, 10⁵ cells/well were cultured on coverslips in a 24-well plate and then treated with doxorubicin (10⁻⁵ M) alone for 6 h, treated with lipid emulsion (0.25%) alone for 7 h, or pretreated with lipid emulsion (0.25%) for 1 h followed by doxorubicin (10⁻⁵ M) for 6 h. After treatment, a TUNEL assay was performed using a TUNEL kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions, and cells were counterstained with 4′,6-diamidino-2-phenylindole (Sigma Chemical Company, St. Louis, MO, USA). TUNEL-positive cells emitting fluorescence were visualized under a Fluoview 500 fluorescence microscope (Olympus, Tokyo, Japan). Three nonoverlapping fields per coverslip were counted per treatment, and the number of TUNEL-positive cells was calculated.