Biolite microwell plate

Cell viability in varying concentrations of the Au@peptide NPs was assessed using PrestoBlue cell viability assay. Human breast adenocarcinoma (MDA-MB-231) and lung fibroblast (IMR-90) cells were seeded in a 96-well flat bottom microplate (BioLite Microwell Plate, Fisher Scientific, Waltham, MA). For IMR-90, 6.0 × 10³ cells per well were plated, and for MDA-MB-231, 5.6 × 10³ cells per well were plated in 100 μL of DMEM culture media. The cells were allowed to grow for 24 h at 37 °C and 5% CO₂ in a humidified incubator. At confluency, cells were dosed with 0, 1, 2, 5, 10, or 50 nM of Au@peptide NPs (in triplicate). Following the administration of the NPs, cells were incubated for 48 h at 37 °C under 5% CO₂. After each period of incubation, PrestoBlue (Life Technologies, Carlsbad, CA) was used as an indicator of cellular toxicity; 11 μL of PrestoBlue was added to each well and incubated for 1 h at 37 °C under 5% CO₂. The 96-well plate was then analyzed using a multimode plate-reader BioTek Microplate Reader (BioTek U.S., Winooski, VT) at 560 / 590 nm wavelength. The percentage of surviving cells was calculated as a normalized ratio of the fluorescence intensity between cells treated with AuNPs and media alone.

Cells were seeded at a concentration of 5000 cells/90 μl per well of either RPMI or DMEM without phenol red and without antibiotics, supplemented with 10% FBS and 2 mM L-glutamine into tissue culture grade 96-well flat bottom microplates (BioLite Microwell Plate, Fisher Scientific, Waltham, Massachusetts, USA) and grown for 24 h at 37°C in a humidified incubator. Afterwards, the intermediate dilutions of the compounds were added to the wells (10 μL) to obtain a final concentration ranging from 0.1 to 200 μM, and the cells were incubated for 24 h or 72 h. Following 24 h or 72 h drug exposure, 50 μL per well of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (Roche Diagnostics, Indianapolis, Indiana, USA) labelling mixture was added to the cells at a final concentration of 0.3 mg/ml and incubated for 4 h at 37°C in a humidified incubator. The optical absorbance of each well in a 96-well plate was quantified using BioTek ELx808 absorbance microplate reader (BioTek Winooski, VT) set at 450 nm wavelength. The percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. The IC₅₀ value was calculated as the concentration reducing the proliferation of the cells by 50% and is presented as a mean (± S.E.) of at least two independent experiments each with triplicate measurements.