Alamar blue

Human MG63 cell line (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Minimum Essential Medium (Sigma-Aldrich, St. Louise, MO, USA) containing 10% fetal bovine serum, 1% antibiotic-antimycotic solution, and 1% GlutaMAX (all from Gibco, Life Technologies Co., Grand Island, NY, USA) at 37 °C and 5% CO₂.
For Alamar Blue assay, 10⁵ cells/well were placed in a 24-well cell culture plate and were left to attach for 24 h. After attachment media were replaced with fresh medium, MPGA/PGA-MNP NCHGs (2 mm × 5 mm gels were used) were submerged using Millipore 24 Well Millicell hanging cell culture inserts 0.4 µm PET (Millipore Co., Billerica, MA, USA), and incubated at 37 °C in a CO₂ incubator. MG63 cells grown in the absence of hydrogel samples were used as control. After 1, 3, and 7 days, media were replaced with 10 times diluted Alamar Blue reagent (Invitrogen, Life Technologies Co., Eugene, OR, USA), then after 2 h of incubation at 37 °C and 5% CO₂, the fluorescence of the samples was measured using a microplate reader (HIDEX Sense, Turku, Finland). Hydrogels were removed before and were placed back after the measurements.

To investigate the in vitro toxicity of the samples, the test was carried out with the cell lines MB49 (tumor cell) and BALB/3T3 (non-tumor cell). Both cell lines were plated on 2 96-well culture plates (Corning Incorporated, NY, USA) to a concentration of 1.10⁵ cell/well in DMEM medium (SIGMA-ALDRICH, USA) in the presence of L-glutamine (2 mmol L⁻¹) and penicillin/streptomycin (100 U mL⁻¹) with the addition of fetal bovine serum (10% SFB) and kept overnight. The cells were exposed to α-Ag₂WO₄ samples irradiated by different sources at the following concentrations: 4.63, 11.58, 23.16 and 46.31 µM and a negative control. After the 24 h exposure, the supernatant containing the nanomaterials was collected and the resulting cells were washed with PBS (1X) buffer and the cell viability test was performed by the resazurin assay-Alamar Blue (SIGMA-ALDRICH, USA), was added into each well and the fluorescence signals were measured using a Fluoroskan (Fluoroskan Ascent FL; Thermo Scientific; Waltham, MA, USA), according to the instructions provided by the supplier. The readings were taken using a spectrophotometer with absorbance in the range 570–600 nm. The experiment was performed in triplicate.

Cell viability was measured using the AlamarBlue (resazurin) fluorescence assay, as described in previous publications (28 (link)). Approximately 5,000 cells were plated in each well of a 96-well plate. The next day the cells were treated with a range of drug concentrations for 72 hours. Fluorescence readings were then obtained after adding AlamarBlue (Sigma) using a FLUOstar Omega microplate reader (BMG Labtech). IC₅₀ values were calculated using log-transformed and normalized data (GraphPad Prism 9).

The viability and proliferative activity of cells cultured on biomaterials and without the biomaterial were evaluated using a resazurin-based indicator of cell metabolic activity (Alamar Blue, Sigma). Measurements were performed at day one, two, and five in both cell lines. For experiment, cell media were collected and replaced with fresh medium containing 10% of resazurin dye. Cultures were incubated for 2 h at 37 °C, followed by the collection of media. Collected media were undertaken for spectrophotometric measurements by means of microplate reader (BMG Labtech, Ortenberg, Germany). Absorbance was measured at 600 nm, with the subtraction of plate background absorbance at 690 nm. A decrease in absorbance in all analyzed groups, in relation to not incubated blank sample, was compared to the standard curve to determine viable cell numbers. These values were used for the calculation of the population doubling time parameter, using an algorithm available online [25 ]. After the incubation, cells on materials and in the control were fixed in 4% paraformaldehyde for fluorescence microscopy or in 2.5% glutaraldehyde for electron microscopy.

500 μl of fibroblast suspension (1 × 10⁴ cells mL⁻¹) was seeded on different samples and cultured with or without NIR irradiation for 1, 3 and 7 days. At the prescribed time point, viability of cells was assessed by Alamar Blue (Sigma, USA) assay according to the instructions and the absorbance was measured by a Multiscan Spectrum (Multiskan FC, Thermo, America). The cell statuses were also evaluated by Live/dead viability/cytotoxicity kit (Invitrogen, France) after 1 and 3 days of incubation according to the User Instruction, and epifiuorescence images were obtained by an Olympus BX52 microscope (SMZ745T, Nikon, Japan). The cell morphologies on different samples after incubation for 1 day were also evaluated by fluorescence staining of actin cytoskeleton, focal adhesion, and nuclei according to the operation manual as detailed elsewhere [44 (link)] and observed by an epifiuorescence (SMZ745T, Nikon, Japan).