Spark multimode

PANC1 cells were cultured as previously described [51 (link)]. The IC₅₀ values of human pancreatic cancer cells treated with Blank BM, erlotinib (used as a positive control [52 (link)]), pure ICA (ICA-Raw), or ICA-BM for 24 h, were measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as previously described [53 (link)]. A 96-well culture plate was used in which PANC1 cells (1 × 10⁵ cells) were seeded and incubated in a humified condition (5% CO₂, 37 °C) to confirm the absolute attachment of the cells [54 (link)]. At the end of the 24 h treatment, the MTT protocol was applied and the absorbance at 569 nm was read by using a microplate reader (Spark^® multimode, Tecan Group Ltd., Seestrasse, Maennedorf, Switzerland). The IC₅₀ for Blank BM, erlotinib, ICA-Raw, or ICA-BM was calculated based on the curves obtained measuring the variation of cell viability (%) as a function of increasing concentrations (0.39, 1.56, 6.25, 25, and 100 µM) of Blank BM, erlotinib, ICA-Raw, or ICA-BM.

The MTT viability assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was conducted in order to obtain the IC₅₀ values of untreated A549 cells (control) after treatment with blank-INVA (INVA-APA), 2ME, or 2ME-INVA-APA for 48 h (Caruso et al., 2017 (link)). Then, in a 96-well plate, A549 cells (1 × 10⁵ cells) were seeded and incubated overnight. Afterwards, these were treated with the plain (INVA-APA), 2ME, or 2ME-INVA-APA at (0.39 μM, 1.56 μM, 6.26 μM, 25 μM, and 100 μM). After 48 hours, the utilized medium was changed to solution (MTT) with the concentration of 2 mg/mL, and then prepared plates were placed in an incubator for 4 h at 37 °C. After that, 200 µL of 100% DMSO was added in order to eliminate the formazan, and then the plates were incubated in a 5% CO₂ at 37 °C for 5 min. Then, a microplate reader (Spark^® multimode, Tecan Group Ltd., Maennedorf, Switzerland) identified an absorbance at 569 nm in each well. The results were presented in terms of percent cell viability relative to the control. Following the plotting of dose response curves, GraphPad Prism (GraphPad, Inc., La Jolla, CA), was used to define the IC₅₀ value for each of the experimental condition.

The IC₅₀ values of untreated A549 cells (control) or treated with blank GNE, CTD, or CTD-GNE for 24 h were obtained using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay, as previously described [23 (link)]. Briefly, A549 cells (1 × 10⁵ cells) were seeded into a 96-well plate and incubated overnight for complete attachment. Later, the cells were treated with the blank GNE, CTD, or CTD-GNE at 0.39 µM, 1.56 uM, 6.26 uM, 25 uM, and 100 uM. After 24 h, the medium was replaced with MTT solution (2 mg/mL), and the plates were incubated at 37 °C for 4 h. The purple formazan product was dissolved by the addition of 200 µL of 100% DMSO, and the plates were then incubated for 5 min at 37 °C in a 5% CO₂ incubator. The absorbance at 569 nm in each well was read by using a microplate reader (Spark^® multimode, Tecan Group Ltd., Seestrasse, Maennedorf, Switzerland). The results were expressed as the percent of cell viability relative to the control. Dose response curves were plotted and IC₅₀ values. The IC₅₀ for each of the experimental conditions was calculated using GraphPad prism software (GraphPad, Inc., La Jolla, CA, USA).