Lps ek ultrapure

The following TLR ligands (InvivoGen, each 1 µg/mL) were added into wells of 24-well plate containing 1-2 x 10⁶ PBMCs: Poly(I:C) HMW (TLR3), LPS-EK Ultrapure (lipopolysaccharide, TLR4), Imiquimod (IMQ) (R837, TLR7), Resiquimod (R848, TLR7/8), TL8-506 (TLR8), ssRNA40/LyoVec (TLR8) and CpG-ODN (TLR9). The concentration of 1 µg/mL is within the range of manufacturer recommendation and induced maximum cytokine response (not shown) (10 (link), 12 (link)). Golgi plug (1 µl/mL, BD Biosciences) was added to block the cytokines release and cell culture continued for 18h. For some experiments, PBMCs were stimulated with agonists alone or in combination with recombinant human HBsAg subtype adw (10 µg/mL, Fitzgerld) (15 (link)) and PepMix HBV (LEP) Ultra (2 µg/mL, JPT) and cultured for 5 days at 37°C incubator. In experiments for transcription factor induction, cells were re-stimulated with Phorbol-12-myristat-13-acetate (PMA, 50ng/mL) and Ionomycin (Ion, 1 µg/mL) on day 4.

MoDCs were added to 48 well tissue culture plates (10⁵ per well), coated with different doses of LPS-free HMW-2 (10, 1 and 0.1 µg/ml) for 16 h. Cultures were incubated for 2 h and then treated for 4 h with ultrapure E. coli LPS (100 and 10 ng/ml, LPS-100, and LPS-10, LPS-EK Ultrapure from Escherichia coli Invivogen, cat # tlrl-peklps) and supernatants collected for cytokine quantification. Controls were moDCs incubated only with LPS-free HMW-2. Cytokines were quantified using DuoSet ELISA (R&D Systems) and Magnetic luminex assay kits (R&D Systems).

BMDMs were plated non-TC treated multi-well plates (Falcon, Agawam, MA, USA) at a density of 100,000 cells/cm² and cultured in BMDM media + 20 ng/mL M-CSF. For polarization, cells were stimulated for 24 h with 100 ng/mL lipopolysaccharide (LPS) (LPS-EK Ultrapure, InvivoGen, San Diego, CA, USA) and 10 ng/mL IFN-γ (Peprotech, Rocky Hill, NJ, USA) for M1 profile, or 20 ng/mL IL-4 (Peprotech, Rocky Hill, NJ, USA) for M2 profile.

Bone marrow-derived macrophages were seeded at 50,000 cells per well in BMDM media in tissue-culture coated 96-well plates (Corning, 3596). The next day, cells were pretreated with 200 ng mL⁻¹ of ultrapure E. coli K12 LPS (InvivoGen, LPS-EK Ultrapure) in BMDM media for 3 h. Cells were then washed once with warm PBS and OPTI-MEM containing 40 μM concentration of chemicals from LOPAC library in a 50 μL volume were applied to BMDM for 6 h. For controls, nigericin at 10 μM was applied to control wells of each plate. IL-1β in the supernatants were detected using a mouse IL-1β ELISA kit (Thermo, 88-7013-77) according to manufacturer’s protocol. IL-1β release level of each plate was normalized to within plate nigericin control (set at 2 ng mL⁻¹) as the screen was performed using three different batches of BMDM. All measurements involving BMDM described in the methods include a 3-hour incubation with LPS (‘priming’).

Rabbit anti-human Carcinoembryonic Antigen polyclonal antibody (Ab) (Dako, Denmark Ab A0115); rabbit IgG control (Vector, CA, USA); PE-conjugated anti-CEACAM1 monoclonal antibody (mAb) (R and D Systems MAb2244); FITC-conjugated mouse anti-human CD14 mAb, human FcR Blocking Reagent, CD14+/CD15+ Microbeads and LS columns (Miltenyi biotec, Germany); PE-conjugated mouse anti-human CD86 mAb and PE-Cy7-conjugated mouse anti-human CD80 mAb (Biolegend, CA, USA); PE-conjugated mouse anti-human CD206 mAb and FITC-conjugated mouse anti-human CD15 mAb (Becton Dickinson, CA, USA); PE-conjugated goat anti-rabbit IgG antibody, endotoxin-free water and Histopaque-1077 (Sigma, USA); fixable Vital dye eFluor 780 (ebioscience, CA, USA); LPS-EK Ultrapure (Invivogen, CA, USA); Ni-NTA agarose and polypropylene column (QIAGEN); Pierce BCA Protein Assay Kit and Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific); Proteome Profiler kits (R&D systems, UK). Antiserum to recombinant rD-7 was raised in mice and has been described previously [29] (link).