Dimethyl sulfoxide (dmso)

2,2-Diphenyl-1-picrylhydrazyl (DPPH, 98%), Fluconazole, Ascorbic acid, methanol, Ammonium molybdate, Sodium phosphate, Conc. H₂SO₄, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 97.5%), ethyl (dimethyl aminopropyl) carbodiimide (EDC, 99%), Folic acid (> 98%), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS, 97%) and Dimethyl sulfoxide (DMSO) were purchased from SRL Chemicals, India. All the cell culture consumables were purchased from HiMedia, India unless and otherwise mentioned. MilliQ water was utilized for all the experiments unless and otherwise mentioned.

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).