Sodium hydroxide solution

On the day after irradiation, the medium was changed to fresh medium containing ¹⁸F-FLT (2.0 mL), with the radioactivity set to 1.0 MBq/flask. The flasks were incubated at 37°C under 20% O₂ and 5.0% CO₂ for 1 h, and then, the medium was removed. The cells were washed with ice-cold PBS (2.0 mL) three times, after which 0.1 mol/L sodium hydroxide solution (2.0 mL, Nacalai Tesque) was added, and the cells were lysed by pipetting thoroughly. The radioactivities of the cell lysate (0.20 mL, n = 3) and ¹⁸F-FLT-containing medium used for the cell uptake study as controls were measured using the Wallac 1480 Wizard 3 gamma counter (Perkin Elmer, MA, USA). In parallel, the protein concentration in the cell lysate was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and then measuring the absorbance at 562 nm using the SpectraMax M5 plate reader (Molecular Devices, CA, USA); a calibration curve was prepared from albumin standards. The percentage of ¹⁸F-FLT cell uptake was normalized to the protein amount (mg).

Compounds were dissolved in dimethyl sulfoxide. Before stimulation with compound, Human TLR7 reporter cell line (Novus Biological) was seeded in 96-well microtiter plate (AGC techno glass) at 5 × 10⁴ cells/well in DMEM (GIBCO) with 10% fetal bovine serum (GIBCO), and 10 ug/ml Blastcidin S HCl (GIBCO) and incubated at 37 °C in 5% CO₂. The plates were incubated over-night and then stimulated with various concentrations of compound and R848. The plates were then incubated an additional 20 ± 1 h at 37 °C in 5% CO₂ and then the secreted embryonic alkaline phosphatase (SEAP) activity was measured as activation of human TLR7. The SEAP activity was evaluated as follows: pNPP (Invitrogen, USA) was added to the incubated sample (50 μl/well); after 15 min, 4 M sodium hydroxide solution (nacalai tesque, Japan) was added thereto (50 μl/well) to quench the reaction; and the absorbance of each sample was measured at 405 nm with microplate reader SpectraMax M4 (Molecular Devices, USA). The 50 % effective concentration (EC₅₀ value) of each compound was calculated based on 100 % of the SEAP activity wherein the test sample comprises no compound.