Multimode synergy ht microplate reader

The effect of self-assemblies in cell metabolism was determined by the Blue Cell Viability assay (Abnova, Taiwan), which is based on the reduction of a dye to a fluorescent compound mediated by metabolically active cells. For that purpose, cells were incubated with self-assemblies (0.01–0.4 mg mL⁻¹) for 24 h. Then, the reagent was added (10%) and incubated for 4 h. After that, fluorescence was read at 535/590 nm (ex/em) in a Multi-mode Synergy HT Microplate Reader (Biotek, US). Cell viability was determined by the interpolation of the emission data obtained from the treated samples and the control ones, which were assigned with 100% viability.

Human dermal fibroblasts, human epidermal keratinocytes (HaCaT), and THP1 human monocytes were used to evaluate the cytotoxic effects of EO-based compounds.
Fibroblasts and HaCaT were routinely grown in high-glucose DMEM supplemented with 10% FBS and antibiotic-antimycotic. Monocytes were cultured in RPMI 1640 supplemented with 10% FBS, 1% HEPES, 1% nonessential amino acids, 0.1% 2-mercaptoethanol 50 mM, 1% sodium pyruvate 100 mM, and antibiotic-antimycotic. Macrophages were obtained by the in vitro differentiation of monocytes by adding 1 µM PMA to the cell culture. All cell types were grown in a humidified atmosphere at 37 °C and 5% CO₂.
The cytotoxicity was determined by measuring cell metabolism through the Blue Cell Viability assay. Cells were seeded on MW96 microplates and incubated with the tested molecules (0.004–0.125 mg/mL) for 24 h. Control samples (not treated and chlorhexidine treated) were also analyzed. Then, the reagent was added (10%) and cells were incubated for 4 h at 37 °C. The reduction of the dye by metabolically active cells was monitored in a microplate reader (Multimode Synergy HT Microplate Reader; Biotek, Winooski, WI, USA) at 535/590 nm ex/em. Cell viability was determined by interpolation of the emission data obtained from the treated samples and the control samples (not treated cells, 100% viability).