Mtt reagent

Aliquots of IDW and CDW fluids were defrosted and diluted (1:4, 1:10 and 1:40 v/v) with culture medium without FBS, and filtered through a 0.22 µm filter (Symta, Madrid, Spain). The cellular toxicity against proliferating Caco-2 cells of the diluted fluids was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, Caco-2 cells, seeded the previous day at a density of 3.6 × 10⁵ cells/mL (100 µL/well), on 96-well plates were incubated with cell culture medium without FBS (control) or with diluted IDW and CDW fluids (1:4, 1:10 and 1:40, v/v) for 4 or 24 h. Then, the supernatants were replaced by MTT reagents (0.5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA). After 3 h of incubation, the MTT reagent was removed from the wells and 100 µL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) was added to dissolve formazan crystals. Absorbance at 570 nm was measured on a Multiskan plate reader (Thermo Scientific, Waltham, MA, USA). Control absorbance was considered 100% cell viability and the results were expressed as the percentage of cell viability relative to untreated control cells. Assays were carried out in triplicate and three independent experiments were performed.

Material cytotoxicity was assessed for the different eluates (1:1, 1:2 and 1:4) of AHP, AHPbcs and ESbcs cultured with hPDLSCs (test groups) and compared with hPDLSCs cultured in unconditioned growth medium (negative control group). This analysis was performed via a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, as previously reported by similar studies (Rathinam et al., 2021 (link)). In brief, hPDLSCs were seeded onto 96‐well plates with 180 μl of DMEM and stored for 24 h at 37°C, 5% CO₂ and 95% humidity. The material eluates were placed in the culture medium with 1 × 10⁴ hPDLSCs (n = 3). An MTT reagent (Sigma Aldrich) was added for 4 h, following its manufacturer's instructions. When a purple precipitate was detectable, Dimethylsulfoxide (DMSO) (Sigma‐Aldrich) was added to each well (100 μl/well), and plates were covered and kept in dark conditions for 4 h to solubilize the formazan crystals produced by viable cells, after reducing the MTT reagent. After 24, 48 and 72 h of culture, light absorbance per well was recorded by means of a microplate reader (ELx800; Bio‐Tek Instruments) at 570 nm wavelength. Culture media with fresh eluates from the respective groups were replaced every 3 days.

TSGH8301 and T24 human bladder cancer cells were seeded in 96-well plates at a density of 2 × 10³ cells (TSGH8301) or 1 × 10³ cells (T24) per well. Doxycycline (1 μg/mL) was added to the FXR overexpression groups for 72 and 96 h. Next, MTT reagent (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma-Aldrich, Brulington, MA, USA) was added, and after 3 h of incubation, MTT reagent was removed and cells were lysed and measured at an absorbance of 590 nm.

1 × 10⁴ cells were seeded and treated for 72 hours with different concentrations of either a BRAF inhibitor (PLX4032, LGX818 or GSK2118436), a MEK inhibitor (MEK162), an ERK inhibitor (SCH772984). DMSO treatment was used as a control. After 72 hours, the medium was removed and fresh RPMI1640 supplemented with 10% FCS and 8% MTT reagent (Sigma, 5 mg/ml in PBS) was added, and the cells were incubated at 37°C. After 1 hour, the RPMI1640 with MTT reagent was removed and 10% SDS (Sigma) and 95% isopropanol/ 5% Formic Acid (Sigma) (ratio 1:1) were added. After 5 min of incubation at 37°C, absorbance was measured at 595 nm (reference 620 nm) using a microplate reader.