Colibri 2 led illumination

The changes in the size and shape of the MCF7 spheroids were monitored using an inverted optical Axio Observer.Z1 microscope (Zeiss, Göttingen, Germany) in a bright field. Morphological changes after treatments were observed at 0, 24, and 48 h. In addition, the qualitative differences in the viability of cells or spheroids subjected to different treatments were investigated using a live/dead assay kit (Invitrogen, Carlsbad, CA, USA). Samples were prepared according to the manufacturer’s instructions and imaged under fluorescence illumination using a Colibri.2 LED illumination and an Apotome.2 system (Zeiss) coupled with an Axio Observer.Z1 microscope (Zeiss). LED illuminator intensity and exposure time were specifically set at 30% and 50 ms for FITC and 60% and 800 ms for EtHD1 channels. Micrographs were obtained using a 10× objective.

Cell morphology was assessed by actin/DAPI staining. The samples were washed three times with PBS to remove the remaining culture medium, and the cells were fixed with paraformaldehyde (4%, v/v) for 30 min at room temperature. The cell membrane was permeabilized by incubating the samples in 0.1% Triton X-100-PBS solution for 5 min. The filamentous F-actin in the samples was stained with 1X Phalloidin iFluor 647 and nuclei were stained with a 1 μg/ml DAPI solution in PBS for 90 min at room temperature. The stained samples were observed using an Axio Observer. Z1 microscope (Zeiss, Jena, Germany) equipped with Colibri.2 LED illumination and an Apotome.2 system (Zeiss, Jena, Germany).

The LIVE/DEAD^® assays (Invitrogen) were conducted according to the manufacturer’s instructions. Briefly, the culture medium was removed from cells or printed constructs and replaced by HEPES (2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid). The LIVE/DEAD^® reagent was then added to the samples and incubated for 20 min at room temperature, followed by three washes with PBS. Fluorescence images were obtained using an Axio Observer. Z1 microscope (Zeiss, Jena, Germany) equipped with Colibri.2 LED illumination and an Apotome.2 system (Zeiss, Jena, Germany).