Varioskan flash device

The PDELight HTS cAMP phosphodiesterase kit (Lonza, LT07-600) was used to determine PDE activity, per operated according to the manufacturer’s instructions (Protocol B). One microliter of PDE eluate expressed in eukaryotes was incubated with osthole for 20 min in PDE buffer (10 mM Tris-HCl, pH = 7.5). Then, 2 μM of cAMP was added, and the solution was kept under incubation for 60 minutes. Then, 10 μl of the stop solution was added to the reaction to stop further phosphodiesterase activity, and 20 μl of the reconstituted AMP detection reagent was then added to the wells followed by incubate for 10 minutes at room temperature, luminescence was measured using a Varioskan Flash device (Thermo Fisher Scientific) after 10 minutes later.

Human Embryonic Kidney 293 Blue (HEK-Blue) SEAP reporter cells overexpressing murine TLR7, human TLR7, or human TLR8 were used in activation assays. The parental control cell lines HEK-Blue Null2-k, Null1-k and Null1 were used as control. All cell lines were purchased from InvivoGen (San Diego, CA, USA). Cells were seeded into 96-well plates (5 × 10⁴/well). After 24 h, cells were transfected with the synthetic oligonucleotides (5 µg/ml) or control oligonucleotide complexed to the transfection agent LyoVec (InvivoGen #LYEC-RNA, San Diego, CA, USA) according to the manufacturer’s instructions. Cells were stimulated with indicated agents dissolved in HEK-Blue detection reagent (InvivoGen #hb-det2, San Diego, CA, USA). Each condition was performed in duplicate. The reporter protein SEAP was detected using the Varioskan Flash device (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of OD 655 nm.