Mtt dye

The cytotoxic effect of lipopolysaccharide (LPS), as well as EPS400, on macrophage cells was tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [22 (link)]. Briefly, macrophages were seeded at a concentration of 2 × 10⁵ cells/well in 96-well plates (Thermo Scientific, Santa Clara, USA). After 2 h of incubation, varying concentrations of LPS (0.001, 0.01, 0.1, 1, and 10 µg/mL) (Sigma-Aldrich, USA) were added and incubated for 24 and 48 h to optimize the concentration of LPS, whereas varying concentrations of EPS400 (12.5, 25, 50,100, 200, 400, and 800 μg/mL) were added and incubated for 24 h to evaluate the cytotoxicity of the EPS400. Then, 10 μL of MTT dye (5 mg/mL concentration) (Himedia, India) and 90 μL RPMI medium were added to each well and incubated at 37 °C for an additional 4 h. The medium was removed using centrifugation at 3000× g at 4 °C for 10 min and 150 µL of dimethyl sulfoxide (DMSO) (SRL chemicals, Mumbai, India) was added to solubilize the formazan crystals. The absorbance of each well was determined at 570 nm using a microplate reader (BioTeK, USA).

Cucurbitacin-B, Hoechst 33342, capase-8, -9, and -3 inhibitors (ZIETD-FMK (caspase-8), Z-LEHD-FMK (caspase-9), and Z-DEVD-FMK (caspase-3)) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were commercially obtained from Sigma-Aldrich (St. Louis, MI, USA). Caspase-8, -9 and -3 activity kits were obtained from BioVision, Milpitas, CA, USA. All Ham’s F12K, fetal bovine serum and antibiotic–antimycotic solutions used for culturing PC-3 cells were obtained from Gibco (ThermoFischer Scientific, Waltham, MA, USA). MTT dye, NAC or N-Acetyl cysteine and the total RNA purification kit were procured from HiMedia Labs, Mumbai, India. The cDNA synthesis and qPCR kit (Verso cDNA synthesis) and the DyNAmoColorFlash SYBR Green kit were procured from ThermoFischer Scientific, USA.

Cucurbitacin-B (SKU: PHL82226) and inhibitors of respective caspases, propidium iodide (PI), DCFH-DA, and Hoechst-33342 dye were commercially procured from Sigma-Aldrich (St. Louis, Missouri, USA). The caspase-specific kits used in the study were procured from BioVision, Waltham, Massachusetts, United States. Materials required for tissue culture including Ham’s F12K media, FBS along with antibiotic–antimycotic solution, SYBR Green kit (DyNAmo ColorFlash), and Verso cDNA Synthesis Kit were obtained from Thermo Fisher Scientific, Waltham, Massachusetts, USA. MTT dye along with the total RNA purification kit was obtained from HiMedia Labs, Pune, India.