Raw blue reporter cells

Human TLR7, mouse TLR7 HEK293, and RAW-Blue reporter cells were purchased from InvivoGen and were cultured following the vendor’s instructions. Murine BMDCs were harvested using a previous protocol (68 (link)). Briefly, bone marrow harvested cells from 8-week-old female C57BL/6J mice were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), penicillin and streptomycin (P/S; 100 U ml⁻¹), 50 μM β-mercaptoethanol, Flt3L (600 ng/ml), and granulocyte-macrophage colony-stimulating factor (5 ng/ml). The medium was changed on day 5 and on day 9; nonadherent cells were harvested, counted, and plated for the assay. MC38 cells were provided by J. Schlom (National Cancer Institute) and cultured in Dulbecco’s modified Eagle’s medium (GE HealthCare Life Sciences) supplemented with 10% FBS and P/S (100 U ml⁻¹). CT-26 cells were purchased from American Type Culture Collection and cultured in RPMI 1640 with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml).

THP1-Xblue-CD14 reporter cells (InvivoGen, France) were cultured and stimulated following manufacturer's instructions. Cells were incubated with 0.1–10 μM FYT21 or 1–40 μM HVF18 and stimulated with 1–1,000 ng ml⁻¹ of P. aeruginosa or E. coli LPS (Sigma), 1 μg ml⁻¹ purified LTA (InvivoGen), 10 μg ml⁻¹ zymosan A (Sigma), 100 ng ml⁻¹ purified flagellin (InvivoGen), 1–100 ng ml⁻¹ recombinant TNF-α (InvivoGen), 0.1% CM of various P. aeruginosa strains or no stimulus. In addition, cells and/or peptide were pretreated with 5–100 μg ml⁻¹ dermatan sulphate (DS36)41 (link). After 18–22 h incubation at 37 °C and 5% CO₂, 20 μl of the supernatants were transferred to new 96-wells plates and 180 μl of QUANTI-Blue (InvivoGen) was added. Plates were incubated at 37 °C and the levels of secreted embryonic alkaline phosphatase (SEAP), which is controlled by the activation of transcription factors NFκB and AP-1, were measured after 1–2 h at an absorbance of 600 nm. To establish the effect of FYT21 on activation of NFκB/AP-1 via TLR9, RAW-Blue reporter cells (InvivoGen) were cultured according to manufacturer's instructions, incubated with 10 μM FYT21 and stimulated with 500 ng ml⁻¹ ODN1826 (InvivoGen).

Human TLR7, mouse TLR7 HEK293, or RAW-Blue reporter cells (InvivoGen) were seeded at 3 × 10⁴ cells/96-well plate for 24 hours and then treated with R848, R848-BPDs, PBS, or dimethyl sulfoxide for 48 hours. To assess innate immune stimulation, secreted alkaline phosphatase was measured from supernatants using QuantiBlue reagent (InvivoGen) to confirm dose-dependent activation of the target TLR receptor. R848-BPDs were confirmed to have low endotoxin levels (<0.1 Endotoxin Units per dose) by the Endosafe Nexgen-PTS system (Charles River).
Alternatively, the immune stimulatory potential of R848 and R848-BPDs in BMDCs was assessed as well. BMDCs were seeded in a 96-well plate (Corning) at 200,000 cells per well. After 24 hours of culture, the medium was replaced with fresh medium or fresh medium with 75 μl of blank polymer (PEG), R848, R848-BPDs or PBS. After 48 hours of incubation, induction of DC maturation by the R848-BPDs was assessed by flow cytometry analysis of the expression of the costimulatory receptor CD86.