Ix81 invert fluorescence microscope

Annexin V staining was accomplished to detect any incidence of apoptosis using a protocol described previously [28 (link)]. Briefly, after washing (3×) with PBS, the treated cells were detached by tripsinization and 1.0 × 10⁶ cells were incubated with 100 μL of 1X binding buffer containing 5 μL FITC-labeled annexin V at 37°C in the dark for 10 min. The cells were then exposed to 200 μL PBS with 5 μL PI. After washing (3×) with PBS, the cells were subjected to the image acquisition using Olympus IX81 invert fluorescence microscope equipped with Olympus DP70 camera (Olympus Corp., Tokyo, Japan) as well as flow cytometry analysis using FACS-Calibur flow cytometr (Becton Dickinson, San Jose, USA).

The AO-EB assay was conducted to further validate possible occurrence of early and late apoptosis and/or necrosis in the cells treated with MNPs (10.71 μg/mL), MTX (1.60 μg/mL) or FA-MTX-MNPs (12.31 μg/mL). Briefly, after washing (3×) with PBS, 40-50% confluent MCF-7 cells were treated with MNPs, MTX or FA-MTX-MNPs and then subjected to the staining with acridine orange (100 μg/mL) and ethidium bromide (100 μg/mL). The cells were then subjected to light/fluorescence microscopy analyses using Olympus IX81 invert fluorescence microscope equipped with Olympus DP70 camera (Olympus Corp., Tokyo, Japan).