Plan apochromat 10 0.45 objective

The cell viability of MG-63 cell laden hydrogels and 3D spheroid-hydrogel hybrids was accessed by using the AlamarBlue® Cell Viability assay. In brief, MG-63 spheroids and hydrogel-3D spheroid models were incubated with cell culture medium containing 10% (v/v) AlamarBlue® reagent, at specific timepoints according to the manufacturer's instructions. After incubation, the fluorescence of the resorufin product was recorded in a Synergy HTX multi-modal microplate reader (λ ex : 540 nm, λ em : 600 nm), by using a black-clear bottom 96-well plate (Corning, NY, US). Both assays were performed in accordance with the manufacturer's instructions. To further access viability, cell laden hydrogels and 3D spheroid-embedded hydrogels were incubated with Calcein AM and PI (Live/Dead assay), for imaging live and dead cells, respectively. Briefly, samples were labelled with Calcein-AM (5 µg mL -1 , in dPBS) and Propidium Iodide (PI) (5 µg mL -1 in dPBS) for 1 h, at 37 °C. Following the incubation period, the samples were washed three times with dPBS. Bioimaging of Live/Dead labelled 3D spheroids, cell laden hydrogels and hydrogel-3D spheroid models was performed in a Zeiss Imager M2 upright widefield fluorescence microscope equiped with a Plan-Apochromat 10×/0.45 objective. All fluorescence images were acquired and post-processed in Zeiss Zen Software SP 2.1 (Carl Zeiss, Germany).