Viability cytotoxicity assay kit for animal live and dead cells

Cell viability was monitored using the Viability/Cytotoxicity Assay Kit for Animal Live and Dead Cells (#3000, Biotium, CA, USA), according to the manufacturer’s instructions. Scaffolds seeded with hADMSCs were evaluated for cell viability and colonization after 1 week of proliferation in MSC expansion medium. In addition, visualization of the live cells attached to the UP scaffolds was examined after 2, 5 and 8 weeks culture in osteogenic differentiation medium. The constructs were captured as z-stack images using the EZ—C1 3.20 software. For live/dead staining, hADMSCs/scaffold constructs were doubly stained with calcein AM and ethidium homodimer, staining living and dead cells, respectively. For each scaffold, ten serial sections were taken with a step/section set at 50 μm in the Z direction. In total, an area of 500 μm in height was analyzed for each scaffold at each time point, so both cell attachment and penetration along the z-axis could be visualized. Fluorescence observations were performed by a confocal upright fluorescence microscope (Nikon D-Eclipse 80i C1). Quantification of fluorescence intensity was determined using Image J software (NIH, Bethesda, MD, USA). Corrected total cell fluorescence (CTCF) was calculated using the formula CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings) [77 (link)].

Live/Dead cell assay was performed using Viability/Cytotoxicity Assay Kit for Animal Live and Dead Cells (Biotium, Fremont, CA, USA) on the 15th day of the spheroid study for both single and multiple treatment groups according to the manufacturer’s protocol. The assay provides green fluorescent by calcein AM for staining live cells and red fluorescence by ethidium homodimer III (EthD-III) staining the dead cells. The images were captured by an Evos FL fluorescence microscope at 10× magnification (Thermo Fisher Scientific, Waltham, MA, USA) using GFP (green fluorescence protein) and RFP (red fluorescence protein) filters, respectively. The images were analyzed for fluorescence intensity using ImageJ 1.42 software.