Cytotoxicity and Proliferation Assay of NIH3T3 Cells

Toxicity and cell viability were conducted by measuring lactase dehydrogenase (LDH) release and WST-1 assay, respectively. Briefly, NIH3T3 cells were cultured at 10⁴ cells per well in a 96-well glass culture plate that had DMEM accompanied with 10% FBS for 24 h. The prepared films were cut into circular discs of 5 mm diameter and were UV-sterilized for 2 h. After sterilization, the films were placed in the wells of 96-well culture plates that had layers of the cultured NIH3T3 cells. The cells were cultured for another 24 h in contact with the films at 37 °C and under a 5% CO₂ atmosphere. LDH and WST-1 kits were used, in accordance with the protocol of the manufacturer, to measure cytotoxicity and cell viability. The culture medium absorbance for LDH and cell viability was measured at 490 nm and 450 nm, respectively. LDH toxicity was calculated by the following equation.
$$Cytotoxicity \left(\%\right)=\frac{A-C}{B-C}\times 100$$
where A represents the test substance, B represents the highly toxic control (lysis buffer), and C represents the low toxic control (tissue culture plate).
Cell proliferation of NIH3T3 cells cultured with the prepared film was determined by WST-1 assay on day 1, day 3, and day 7. NIH3T3 cells were cultured using DMEM with 10% FBS at 37 °C under a 5% CO₂ atmosphere. The treatment medium was replaced every other day. On each predetermined day, WST-1 was added to the culture plate and further incubated for 4 h. Finally, the medium was transferred to a new 96-well plate, the absorbance of the medium was measured at 450 nm by SPECTROstar ^® nano microplate reader, BMG Labtech, Offenburg, Germany. For LDH toxicity, cell viability, and cell proliferation, the sample size was 3 (n = 3).