Tlrl pm2s 1

HEK-Blue hTLR cells lines were seeded in 384-well plate (4 × 10⁴ cells per well) with 20 μL DMEM medium (containing 10% heat-inactivated FBS), and cultured with 20 μL of DMEM medium containing indicated concentrations compounds with or without TLR-specific agonists at 37 °C in a 5% CO₂ humidified incubator for 24 h. After incubation, another 40 μL of Quanti-Blue solution (req-qb12, Invivogen) was added and incubated for 30 min. Absorbance was measured at 620 nm using a microplate reader. The IC₅₀ of compound was calculated and processed using Origin 9.0 software. TLR-specific agonists to selectively activate respective TLRs: Poly I:C, lipopolysaccharide (100 ng/mL, tlrl-b5lps, Invivogen), Pam₃CSK₄ (200 ng/mL, tlrl-pms, Invivogen), Pam₂CSK₄ (200 ng/mL, tlrl-pm2s-1Invivogen) and R848 (5 μg/mL, tlrl-r848, Invivogen) were used. Value of IC₅₀ were calculated and fitted using Origin 9.0 software.