Figure 8

Acridine orange – ethidium bromide (AO – EB) assay for detection of apoptotic and/or necrotic MCF-7 cells. A) Untreated cells as a negative control. B) Cells treated with the free MNPs (10.71 μg/mL). C) Cells treated with the free MTX (1.60 μg/mL). D) Cells treated with the PEGylated FA-MTX-MNPs (12.31 μg/mL). The cultured cells, at 40-50% confluency, were treated and then subjected to the staining with acridine orange (100 μg/mL) and ethidium bromide (100 μg/mL) followed by light/fluorescence microscopy analyses. The untreated cells (panel A) and the MNPs treated cells (panel B) were viable with impermeable membrane to EB, which showed normal round nuclei stained green. The cells treated with the free MTX or the PEGylated FA-MTX-MNPs (panels C and D, respectively) were apoptotic (stained orange) with condensed and/or fragmented nuclei or necrotic (stained red).