D luciferin salt

THP-1 cells were transiently transfected with luciferase reporter vector with NFκB promoter sequence upstream of the luciferase gene. Transfection was performed following the manufacturer’s instruction in 96 well plates using Lipofectamine 3000 (Invitrogen, USA). Two days after transfection, the experiment was performed as described earlier^{55 (link)}, with some modifications. Used media was replaced with media containing the test compound and control sample. After 1 hour LPS was added at a concentration of 500 ng/ml, where required and incubated further for 12 hours. D-Luciferin salt (Perkin Elmer, USA) at a final concentration of 150 μg/ml was added to the cells and incubated at 37 °C, protected from light. Relative percentage changes in light emission intensity were measured from each well, using Envision microplate reader (Perkin Elmer, USA), LPS induction alone was measured as 100% activity of the NFκB reporter gene^{55 (link)}.

Luciferase reporter vector with NFκB promoter sequence upstream of the luciferase gene was transiently transfected into adherent and differentiated THP-1 cells. Transfection was performed using the Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA), following the manufacturer’s instruction. The experiment was performed as described earlier [45 (link)], with minor modifications. The consumed media was carefully aspirated 48 h post-transfection, and fresh media was added. The experimental wells were treated with different concentrations of WSSO. After 1 h, LPS was added at a final concentration of 500 ng/mL where required and incubated further for 12 h. D-Luciferin salt (Perkin Elmer, USA), at a final concentration of 150 μg/mL, was added to the cells and incubated at 37 °C in the dark. Relative percentage changes in luminance intensity were measured from each well using the Envision microplate reader (Perkin Elmer, USA). Measurements from LPS alone wells were considered as 100% activity of the NFκB reporter gene.

THP-1 cells were transiently transfected with luciferase reporter vector with NF-κB promoter sequence upstream of the luciferase gene. Transfection was performed following the manufacturer’s instruction in 96-well plates using Lipofectamine 3000 (Invitrogen, USA). Two days after transfection, the experiment was performed as described by Ishimoto et al. (2015) (link) with the following modifications. Used media was replaced with media containing test compound and control. After 1 h, LPS was added at a concentration of 500 ng/ml, where required and incubated further for 12 h. D-Luciferin salt (PerkinElmer) at a final concentration of 150 μg/ml was added to the cells and incubated at 37°C, protected from light. Relative percentage changes in light emission intensity were measured from each well and calculated, and LPS alone was measured as 100% activity of the NF-κB reporter gene.