Human duo set kit

The inhibition potential of compounds 1–14 against cytokine (TNF-α) production in human leukemia cell line (THP-1) was evaluated by applying the reported protocol [30] (link). In this assay, THP-1 cell line from the ECCC (UK) were grown, and maintained in the RPMI-1640, comprising mercaptoethanol (50 µM), sodium pyruvate (1 mM), FBS (10%), glucose (5.5 mM), L-glutamine (2 mM), and HEPES (10 mM). At confluency of 70%, 2.5 × 10⁵ cells/mL were plated in 24- well plates of cell culture. PMA (phorbol myristate acetate) (20 ng/mL) was added to differentiate them into the macrophage mimicking cells, and incubated for 24 h in the presence of 5% CO₂ at 37 °C. Lipopolysaccharide B of Escherichia coli (50 ng/mL) was used to stimulate the culture, assessed with compounds 1–14, and placed for 240 min in 5% CO₂ at 37 °C. TNF-α level in the supernatants was determined on ELISA through human Duo Set kit (R & D Systems) (USA), according to instructions of manufacturer.