Alamarblue viability assay

Cell viability was assessed using two fluorescent indicator dyes: alamarBlue viability assay (Invitrogen) and via 5‐carboxyfluorescein diacetate (5‐CFDA‐AM; Invitrogen).⁴⁰Resazurin is the active component of the alamarBlue assay and is a nontoxic compound reduced by live cells and is an effective measure of cellular metabolism. Following incubation of the cells, cells+collagen constructs were washed twice with DPBS, and a 5% vol/vol alamarBlue solution in DPBS was added to the constructs. Constructs were then incubated at 37°C for 1 and 2 h in the dark and fluorescence was measured using a Synergy HT plate reader (BioTek) by transferring 50 μl of the media into a new 96 well culture plate.
To study cell membrane integrity of the AF cells encapsulated in 3D collagen, 5‐CFDA‐AM was diluted in DPBS and constructs were incubated with the diluted solution for 30 minutes at 37°C. Images of cells at different locations (including depth) within the constructs were taken by an inverted confocal laser scanning microscope (Olympus Fluoview 1000; Olympus Life Sciences).
Collagen fibers were imaged using the same microscope by confocal reflectance microscopy technique. Samples were illuminated by an Argon laser (λ = 488 nm) and the same wavelength was collected to image the collagen fibers.

Viability testing on 2D cell culture was performed via Alamar Blue viability assay (Thermo Fisher Scientific, Inc.). After treatment with TKI, 10 µl Alamar Blue solution was added to each well, according to the manufacturer's instructions. Absorption was measured after 3 h using an Infinite M Plex Microplate Reader (Tecan Trading AG).