Alamar blue assay

Cell viability was measured using alamarBlue assay according to the manufacturer's recommendations (AbD Serotec, Raleigh, NC, USA). In brief, we cultured cells in 96-well plates in 100 μl of the appropriate medium and at the indicated time point, and 10 μl of alamarBlue substrate was added and plates were incubated in the dark at 37 °C for 1h. Reading was subsequently taken using fluorescent mode (Ex 530 nm/Em 590 nm) using BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, USA).

The cells (5 × 10⁴ cells/mL) were seeded on the sample surfaces and cultured for 1, 3, and 5 days. At each time point, the samples were rinsed with PBS and moved to a new 24-well plate. Immediately, 0.5 mL of AlamarBlue assay (AbD Serotec Ltd., Kidlington, UK; diluted 10-fold with culture medium was added to each well and cultured for another 2 h. Soon after that, 100 μL culture medium from each well was transferred to a black 96-well plate and measured using an enzyme-labeling instrument (BIO-TEK, ELX 800) with an absorption wavelength of 560 nm and a scattering wavelength of 590 nm).

Periodontal ligament stem cells (PDLSCs), at the fourth passage, were seeded in a 48 multi-well plate using a density of 1.0 × 10⁴ cells. To evaluate the proliferation and viability of magnetically stimulated PDLSCs (PDLSCs MAG) and unstimulated PDLSCs, the Alamar Blue assay (AbD Serotec Ltd., Kidlington, UK) was used. At 4, 7, 14 and 21 days after cell seeding, the cells were rinsed with PBS (Sigma Aldrich, Milan, Italy) and 200 µL of DMEM without phenol red (HyClone, Cramlington, UK) containing 10% (v/v) Alamar Blue was added to each sample. The samples were incubated for 4 h in a 5% CO₂ diluted atmosphere at 37 °C. Subsequently, 100 microliters of the solution were removed from the wells and transferred to a 96-well plate.
The assay was based on a redox reaction in the mitochondria of the cells, and the colored product was transported out of the cell.
The optical density was measured through a spectrophotometer (Sunrise; Tecan, Mannedorf, Zurich, Switzerland) at wavelengths of 570 and 595 nm. The experiments were conducted three times in triplicate.

hMSC-seeded hydrogels were cultured for 0, 1, 3, 7, 14, 21, 28, and 42 days. After the culture period, the hydrogels were washed three times in PBS and analyzed for cell viability and metabolic activity. Cell viability was qualitatively assessed with a LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes, USA) according to the manufacturers’ instructions. Fluorescence confocal microscopy (Leica SP5 inverted microscope, Leica Microsystems, UK) was used to visualize live (calcein; green) and dead (ethidium homodimer-1; red) cells. The metabolic activity of cells in the hydrogels was quantified by the AlamarBlue® assay (Serotec, USA). This assay is based on the fluorescent signal output produced by metabolically active cells. Measurements were made at 570 nm and 600 nm. Cell-free hydrogels and empty wells were used as controls. All data were normalized to DNA content (described in Section 2.11.) at each time point.

Alamar blue assay (AbD Serotec) and Cytotoxicity Detection Kit (LDH) (Roche) were used according to the manufacturer’s protocol. For toxicity testing, C2C12-cells were seeded at a density of 15,000 cells/cm². Adherent cells were incubated for 24 h with a 1:1 mixture of culture medium and the washing solution to test. As controls, a 1:1 mixture of culture medium and 0.1% Triton X-100, respectively, of 0.05% and 0.005% SDS was used. After 24 h, the test medium was probed for LDH, and cells were incubated with the Alamar blue reagent for 3 h. In Alamar blue testing, cells cultivated with fully supplemented cell culture medium (DMEM) were used as positive control. All other groups were drafted as “relative survival” according to the DMEM group. For LDH testing, 0.1% Triton X-100 was used as positive control. This concentration leads to full death of the indicator cells. All other groups were drafted as “relative survival” according to the 0.1% Triton X-100 group.

Cell viability was measured using Alamar blue assay according to the manufacturer's recommendations (AbD Serotec, Raleigh, NC, USA). In brief, to cultured cells in 96-well plates 10 μl of Alamar blue substrate was added and the plates were incubated in the dark at 37 °C for 1 h. Reading was subsequently taken using fluorescent mode (Ex 530 nm/Em 590 nm) using BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, USA).