Cellstar 96 well culture plate

After a total of 24 h from the irradiation, an MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed to evaluate cell viability. Here, 100 µL of MTT (2.5 mg/mL) was added to 100 µL of DMEM without phenol red; the solution was added to each well and left in contact with the cells for 3 h in the incubator. After removing the MTT from the wells, 300 µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, Milan, Italy) were added to lysate the cellular and mitochondrial membranes and solubilize the formazan. Next, 100 µL of each sample were taken and transferred, respectively, to a 96-well plate (Cellstar 96 Well Culture Plate; Greiner Bio-One, G) having an area of 0.28 cm². The absorbance of each well was detected with a spectrophotometric method using an ELISA Plate Reader (iMARK Microplate Absorbance Reader; BioRad, Milan, Italy) at 570 with 690 nm as the reference wavelength. Cell viability was calculated as a percentage ratio of the absorbance of the sample to the absorbance of the control.

Cytotoxicity induced by the membranes was assessed by an MTT test in 96-well plates (Cellstar 96 Well Culture Plate, Greiner Bio-One, Kremsmünster, Austria). Membranes were cut as circles with the correspondent well’s area (0.32 cm²). For comparison, an equivalent amount of LDH and NaNAP samples in powder present in the composites (Table 1) were also evaluated, as well as pristine PEBA membrane. NHDFs were seeded onto membranes in well plates (0.35 × 10⁵ cell/well in 200 µL/well) and incubated for 24 h at 37 °C in humidified atmosphere containing 5% CO₂ (CO₂ Incubator, PBI International, Milano, Italy). Powdered samples were added after cell confluence and then the same MTT procedure was applied for all the samples. Briefly, medium was removed after 24 h of treatment, cells were washed with PBS (10% v/v, 200 µL/well), exposed to 50 µL MTT solution (2.5 mg ml⁻¹ solubilized in DMEM w/o red phenol) diluted in 100 µL of DMEM (w/o red Phenol) and incubated for 3 h at 37 °C. Next, the MTT reagent was removed and 100 µL of DMSO was added into each well to lyse cells. Finally, the absorbance was read at 570 nm with 690 nm as wavelength reference by means of an ELISA plate reader (Imark Absorbance Reader, Biorad, Milan, Italy). Cell viability was calculated as the percentage ratio between the absorbance of each sample and the absorbance of controls (cell substrates in growth medium).