96 well culture microplates

The assay was performed using the colorimetric ECM Cell Adhesion Array Kit (Merck Millipore, Germany) and also 96-well plates coated by ourselves. The ECM Array microtiter plates, pre-coated with different extracellular matrix proteins (Purified human Collagen I, Collagen II, Collagen IV, Fibronectin, Laminin, Tenascin, Vitronectin), were used according to the manufacturer’s instructions. Independently, 96-well culture microplates (Corning, USA) were coated with fibronectin (20 μg/ml) or laminin-1 (10 μg/ml), and incubated at 4 °C overnight. Non-specific binding sites on the plates were blocked by a one-hour incubation with 0.5% BSA in the RPMI medium at 37 °C in a CO₂ incubator. Next, the cells were counted and re-suspended in the culture medium to final concentration. Then, 50 μl of cell suspension of 4 × 10⁵ cells/ml were seeded in the wells (in triplicates) and allowed to adhere for 30–60 min at 37 °C. After that, unattached cells were removed by washing, and adherent cells were fixed with 4% PFA for 15 min and then stained with crystal violet solution (Sigma Aldrich, USA) for 10 min. After extensive washing, the dye was extracted using 2% SDS and quantified by spectrophotometry at 550 nm using a microplate reader (Multiskan RC, Labsystems, Thermo, Finland).

The MCF-7 (human breast adenocarcinoma) cell line was purchased from American Type Culture Collection (ATCC) C (MCF-7 cell line, HTB-22™, ATCC^®, Manassas, VA, USA obtained in 2018). In all experiments were performed below 13 passages and cell line authentication was performed by ATCC using STR profiling. MCF-7 human breast adenocarcinoma was seeded in 96-well culture microplates (Corning Inc., New York, NY, USA) at an initial density of 4 × 10³ cells per well and it was cultured under defined conditions at 37 °C in 5% CO₂ and 95% humidity. DMEM medium (Dulbecco’s Modified Eagle Medium, High Glucose, PAA, Pasching, Austria) was supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAA, Pasching, Austria), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), streptomycin-penicillin (50 IUmL⁻¹; Sigma-Aldrich, St. Louis, MO, USA) and 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA) and was used to maintain cell cultures. The two tested Fe₃O₄-graphene oxide nanocomposites loaded with hydroxycamptothecin (c-GO–Fe₃O₄-HCPT, and nc-GO-Fe₃O₄-HCPT, respectively) were added to cell cultures 24 h after from seeding. The final concentrations of the NPs in the culture medium were: 3.125, 6.25, 12.5, 25.0 and 50.0 µgmL⁻¹, respectively. The MCF-7 cells were exposed to nanomaterials for 48 h. Additionally, the effect of GO–Fe₃O₄ and HCPT not conjugated with NPs were analyzed.

In vitro drug testing was performed as previously reported for other anthelmintic assays (25 (link), 26 (link)). Briefly, T. canis L3 were transferred to 96-well culture microplates (Corning, New York, NY, USA) containing 100 larvae/well in RPMI 1640 medium. The larvae were treated with the N-(4-methoxyphenyl)pentanamide and albendazole (previously dissolved in DMSO) at concentrations of 50, 25, 12.5, 6.24, and 3.1 μM and maintained for 72 h. DMSO was added to the medium of the treated groups and controls in order to obtain a final concentration of 0.2% during the periods of treatment. Each drug concentration was tested in at least triplicate, and the experiments were repeated three times. The viability of larvae was scored immediately after adding the drug (time zero) and after 6, 12, 24, 48, and 72 h using an inverted microscope (INV 100; BEL Engineering, Monza, MB, Italy). Larval motility was scored (effect of ≥60%) as 4 (highly active, using the whole body), 3 (slow motion, using the whole body), 2 (movement with only one part of the body), 1 (immotile but not dead), or 0 (dead). The mortality of T. canis larvae was assessed by trypan blue dye exclusion (26 (link)).