Thermo labsystems multiskan rc microplate reader

The bone marrow-derived macrophages (M0, M1 and M2) were placed in 96-well plates at a density of 0.15 × 10⁵ cells/well. After 24 h, boron carbide preparations (B₄C 1 and B₄C 2) were added at concentrations in the range from 0.1 to 200 µg/ml and incubated for 72 h. Next, cells were incubated with MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5 mg/ml)(Sigma-Aldrich) for 4 h and then lysed overnight in lysis buffer (N,N-dimethylmethanamide, sodium dodecyl sulfate, and water) at 37 °C. The absorbance of the solubilized crystal of formazan was measured at 570 nm using a Thermo Labsystems Multiskan RC microplate reader (Thermo Fisher Scientific Inc.) with Genesis Lite 3.05 Software (Thermo Life Sciences). The half maximal inhibitory concentration (IC₅₀) value was calculated using GraphPad Prism 8 software (GraphPad Software). MTT cell viability assay for lines: RAW264.7, J774A.1 and JAWS II was performed and described in our previous work by Kozień et al. [18 (link)]. MTT assays were performed in two independent experiments, each in triplicate. The IC₅₀ was calculated for each experiment individually and averaged on the graph for two independent experiments.

MC38, RAW 264.7, and HT-29 cells (5 × 10³ cells/100 µL/well) and THP-1 cells (1 × 10⁴ cells/100 µL/well) were seeded in 96-well plates (Corning Costar, Corning, NY, USA). After 24 h, CBN preparations (BN-14, BN-17) were diluted to specific concentrations (range 0.1–400 µg/mL) and added to the plates (100 µL/well). The cells were incubated with compounds for the next 72 h. Afterwards, MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 5 mg/mL) (Sigma–Aldrich, St. Louis, MO, USA) was added to a final concentration of 0.55 mg/mL (25 µL/well) and incubated for 4 h, followed by the addition of 75 µL/well of lysis buffer (N,N-dimethylmethanamide, sodium dodecyl sulfate, and Milli-Q water) and incubation overnight at 37 °C. Finally, the absorbance at 570 nm was measured using a Thermo Labsystems Multiskan RC microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with Genesis Lite 3.05 software (Thermo Life Sciences, Waltham, MA, USA). The 50% inhibition of cell proliferation (IC₅₀) values were calculated using GraphPad Prism 9 software (GraphPad Prism Software version 9.0.0, La Jolla, CA, USA).