Bx51 fluorescent microscope

The intracellular ROS levels were assessed by fluorescence microscopy using two different probes: dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) [11] (link), [12] (link). For ROS assays, MDA-MB-231 and MCF7 cells were seeded on Matrigel™- coated (1/30 dilution in FBS-free medium) dishes and after 24 h, when cells were ~40% confluent they were incubated with vehicle, dox (0.1 µM), MnTnHex-2-PyP (5 µM) or both drugs for 1 or 16 h at 37 °C in FBS-free medium. For the DHR assays, CAT (50 U/mL) was used alone or in combination with both drugs. Cells were then washed with warm PBS and incubated with DHR or DHE (10 µM) in FBS-free medium for 25 min at 37 °C. Cell image acquisition was performed using a wide field BX51 fluorescent Olympus microscope with a 40x objective using a 460–490 nm/<520 nm excitation/emission filter for DHR and a 520–550 nm/<580 nm excitation/emission filter for DHE. Cell fluorescence and area were determined using ImageJ (National Institutes of Health) [13] (link) for a minimum of 45 cells per condition. Three to four independent experiments were performed.

Cell spreading evaluation was performed similarly as previously described by Saraiva et al. [17] (link). MDA-MB-231 and MCF7 cells were detached with trypsin, resuspended in complete medium and incubated for 30 min or 16 h with the appropriate drugs. Cells were seeded in Matrigel™-coated coverslips and left to attach for 20 min, 35 min and 12 h or 3 h and 12 h, for MDA-MB-231 and MCF7, respectively. Cells were fixed with 4% PFA, washed with PBS and the coverslips were mounted in Mowiol 4–88 containing DAPI. Image acquisition was performed with a wide field BX51 fluorescent Olympus microscope with a 40 × objective. Cell area was determined using Image J. Mean cell areas were normalized to the untreated control at 12 h. Three independent experiments were performed for each protocol.

Fluorescent gelatin-coated cover slips were prepared as described by Martin et al. [18] . Briefly, coverslips were coated with thin layers of Oregon Green 488-conjugated gelatin, cross-linked with 0.5% glutaraldehyde for 15 min, incubated for 3 min at room temperature with 5 mg/mL NaBH₄ and washed three times with PBS and incubated for 15 min in 70% ethanol. Cells were seeded on gelatin-coated coverslips at a density of 4 × 10⁴ cells per well in complete DMEM and incubated with the relevant compounds. After 16 h cells were fixed in 4% PFA. Analysis was performed on a wide field BX51 fluorescent Olympus microscope with a 40 × objective. The gelatine degradation percentage (per image) was measured using Image J software and was then normalized to the number of cells to obtain normalized degradation value.