N sim e laser confocal microscope

Cells in collagen scaffolds were imaged by scanning electron microscopy (SEM) and laser confocal microscopy as previously described [34 (link)]. For SEM, the samples were washed in 0.1 m sodium cacodylate buffer pH 7.4 and fixed in 2.5% glutaraldehyde 0.1 m sodium cacodylate buffer for 2 h at 4 °C. Before imaging, samples were dehydrated in a series of ethanol, dried in a dessicator overnight, and sputter‐coated with platinum. The Nova NanoSEM 230 (FEI, Hillsboro, OR, USA) was used to acquire all the images. For confocal microscopy cells were fixed in 4% paraformaldehyde for 20 min and stained with Alexa Fluor™ 546 Phalloidin and DAPI (Thermo Fisher Scientific, Waltham, MA USA) for 1 h at 4 °C. For lysosomes detection, cells were collected by trypsinization for monolayer cultures or by digestion in Collagenase type I (Merck Millipore, Burlington, MA, USA) for 3D culture. Cells were then stained with 75 nm LysoTracker™ Green DND‐26 (Thermo Fisher Scientific) for 30 min at 37 °C and cytospinned onto glass slides. For yH2AX immunofluorescence staining, Phospho‐Histone H2A.X (Ser139) (mAb #9718 Cell Signaling, Beverly, MA, USA) was used (1 : 400) and detected with secondary antibody Alexa Fluor™ 488. Images were acquired with an N‐SIM E laser confocal microscope (Nikon Corporation, Tokyo, Japan) and performed at 20× magnification.

Scaffolds with MDA-MB-231 cells cultured for 7 days in static vs. dynamic condition were processed with the LIVE/DEAD^® Viability/Citotoxicity Kit (LIVE/DEAD^® Viability/Citotoxicity Kit (MP03224, Molecular Probes™, Eugene, OR, USA) following manufacturer’s instructions. Briefly, hydrogels were washed with PBS and incubated for 45 min at room temperature (rt) in 300 µL of staining solution, which contains 2 μM Calcein AM and 4μM Ethidium homodimer-1 diluted in PBS. Scaffolds were cut in half along the transversal plane using a scalpel. Images were acquired as described previously29 (link) using a N-SIM E laser confocal microscope (Nikon Corporation).
Quantitative analysis of cell viability was performed processing images using the ImageJ software in order to calculate the green/red (LIVE/DEAD^®) area referred to the specific spots following the maximum entropy threshold-based image segmentation method. The total area (A) covered by specific spots of interest in the total scaffold slice surface was calculated using the following formula: $$A=np·p{d}^2$$ where np is the number of pixels detected and pd is each pixel’s dimension.
Mean values ± SD were reported and statistical analysis was performed using the Mann-Whitney test by GraphPad Prism 6^® software.