2.4. Cytotoxicity Assessment

Cell proliferation and viability were determined with several cytotoxic methods studying the influence of GPs co-cultured with PAECs for 24 and 48 h. The CyQUANT LDH cytotoxicity assay (ThermoFisher Scientific, Waltham, MA, USA) was one of the methods used to measure the cytotoxicity effect on cells. Briefly, PAECs in different densities (0.5–1 × 10⁴ cells) were incubated overnight in a 96-well plate, and cells were washed and exposed to increasing concentrations of GPs (5–100 μg/mL) for 24 or 48 h. The lactate dehydrogenase (LDH) assay was performed according to the manufacturer’s protocol. Absorbance was measured in a SUNRISE Xfluor4 (TECAN, Männedorf, Switzerland) microplate spectrophotometer at 492 nm. All experiments were performed in triplicates for each sample.

To determine the effect of graphene on the proliferation of PAECs, a WST-1 assay (Sigma, Buenos Aires, Argentina) was used. Cells were added to a 96-well plate and cultured for 24 h. For another 24 to 48 h, the GPs were added to the culture. Then, tetrazolium salt (WST-1) was pipetted and incubated with exposed cells for 4 h. Finally, the colored product was analyzed with an ELISA analyzer (TECAN) at 490 nm. All experiments were performed in triplicate for each set of samples.