2.6 Cellular assays
Live/Dead assay . Viability of spheroids was qualitatively analyzed using a Live/Dead assay kit (Thermo Fisher Scientific, Inc.). The activity of intracellular esterase induces non-fluorescent, cell-permeant calcein acetoxymethyl to become fluorescent, giving the viable spheroids a green fluorescence. Ethidium homodimer enters and binds to nucleic acids only into damaged cell producing a red fluorescence thus indicating dead cells. The assay was performed at three time points to monitor the viability of the spheroids during their growth. In detail, the medium was removed from the plates containing the spheroids-embedded hydrogels. The samples were washed twice with PBS. Then, a phosphate buffer solution containing calcein and ethidium homodimer at the concentration suggested by the supplier company was added to the plate that was kept in incubation at 37 °C for 1 h. Finally, the solution was replaced with fresh PBS prior to image the samples under a Fluorescence Microscope (EVOS FLoid Cell Imaging Station, ThermoFisher, Waltham, MA USA).
Mitochondria toxicity assay. MitoTracker Red from Life Technologies was dissolved in PBS and added to the hydrogel containing the spheroids at the 5th day of growth (working concentration: 250 nM). The samples were kept in incubation for 1 h at 37 °C and then imaged under a Fluorescence Microscope (EVOS FLoid Cell Imaging Station, ThermoFisher, Waltham, MA USA).
Cisplatin treatment. MCF-7 spheroids were grown in 0.25%-0.02% and 0.125%-0.02% A-C hydrogels up to 12 days. Then, a DMSO solution of cisplatin was added to the spheroids-embedded hydrogels to reach a final concentration equal to 100 µM. After 24 h incubation the medium was removed and the hydrogels were carefully rinsed with fresh medium prior to perform the Live/Dead assay (Thermofisher), as already reported.
2.7 Immunofluorescence microscopy analysis. After 5 days of growth within the hydrogels, the spheroids were fixed with ice-cold 4% paraphormaldehyde for 30 minutes. Then they were washed twice with PBS and incubated with a mouse anti E cadherin (E-cad; Santa Cruz, sc-8426) antibody overnight (O.N.), according to the manufacturer’s protocol (1:1000 dilution in PBS). Subsequently, the samples were incubated with an anti-mouse Alexa Fluor 488 (AF488) conjugated secondary antibody (Cell Signaling). The images of fluorescently labelled proteins were captured using a fluorescence microscope (Leica LMD7000, Mannheim, Germany).