Tlrl peklps

CD4⁻ MutuDC2s and MutuDC1s were seeded at a density of 2.5 × 10⁵ cells/cm² in 96-well or 48-well plates and incubated for 24 h with 450 μL/cm² of the following TLR agonists diluted in complete medium: Pam3CSK4 (150 ng/mL, tlrl-pms, InvivoGen), poly(I:C) (8.5 μg/mL, tlrl-pic, InvivoGen), LPS from E. coli (100 ng/mL, tlrl-peklps, InvivoGen), ultrapure flagellin from B. subtilis (100 ng/mL, tlrl-pbsfla, InvivoGen), FSL-1 (100 ng/mL, tlrl-fsl, InvivoGen), Gardiquimod™ (1 μg/mL, tlrl-gdqs, InvivoGen), CpG ODN 1826 (1 μM, TriLink BIOTECHNOLOGIES). In all the experiments each condition was plated in technical triplicate. The supernatants were analyzed by ELISA for the presence of IL-6, IL-10, IL-12/IL-23 p40, IL-12p70, and MCP-1(CCL2) using the following kits according to manufacturer's instructions: Mouse IL-6 ELISA Set (555240, BD Biosciences) or Mouse IL-6 ELISA Ready-SET-Go! (88-7064, eBioscience), Mouse IL-10 ELISA Set (555252, BD Biosciences) or Mouse IL-10 (Interleukin-10) ELISA Ready-SET-Go! (88-7104, eBioscience), Mouse IL-12 (p40) ELISA Set (555165, BD Biosciences), Mouse IL-12 (p70) ELISA Set (555256, BD Biosciences), Mouse CCL2 (MCP-1) ELISA Ready-SET-Go! (88-7391, eBioscience).

Human THP-1, U937, HeLa cells, A549, HCT-116, and mouse TRAMP-C2 cells were purchased from ATCC (USA). A549, HeLa and TRAMP-C2 cells were grown in DMEM medium (Invitrogen, USA), while HCT116 cells were cultured in McCoy’s 5A medium (Invitrogen, USA), and both mediums are supplemented with 10% heat-inactivated FCS (Hyclone, USA) and 1% pen/strep (Invitrogen). THP-1 and U937 cells were cultured in RPMI medium (Hyclone, USA) supplemented with 10% heat-inactivated FCS (Hyclone, USA). All cells were grown at 37°C in a humidified 5% CO2 incubator (Thermo Scientific, USA). THP1 cells were differerentiated in PMA (#tlrl-pma, InvivoGen, USA) and stimulated with 1 μg/ml LPS (#tlrl-peklps, InvivoGen, USA) overnight before fresh cell medium was replaced and the cells were either treated with 50% cancer CM or transfected with 1 μg/ml poly (dA:dT) (#tlrl-patc, InvivoGen, USA) with lyovec transfection reagent. Inhibitors THP1 Cells were primed with 100 ng/ml PMA, followed by LPS (1 μg/ml) for 3 hours prior to treatment 1 hour with 1 μg/ml Poly(dA:dT) complexes (AIM2 inflammasome inducer) or A549 tumor CM in the presence of ODN TTAGGG (A151; AIM2 inhibitor, 0.5 μM) or MCC950 (NLRP3 inhibitor, 0.5 μM). Cell supernatants were detected using ELISA.

Purified flagellin from P. aeruginosa (tlrl-pafla; InvivoGen) and ultrapure LPS from E. coli K-12 (tlrl-peklps; InvivoGen) were resuspended in water. CLI095 (tlrl-cli095; Thermo Fisher Scientific), SB203850 (NC9041893; Fisher), and U0126 (19-147; Sigma) were resuspended in dimethyl sulfoxide.

Human rhabdomyosarcoma (RD) cells (ATCC® CCL-136) and SH-SY5Y cells (Procell, #CL-0208) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Hyclone) with 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were infected with BrCr strain EV-A71 (ATCC® VR-1432™) or neurotropic strain EV-A71 (Xiangyang-Hubei-09, GenBank accession no. JN230523.1) at the multiplicity of infection (MOI) of 2 or transfected with N-terminal GFP-tagged EV-A71 2A expression plasmid and/or bi-cistronic reporter plasmid containing Cap-Rluc-vIRES-Fluc. Plasmid construction, transcription in vitro and transfection using Lipofectamine 2000 reagent (Life Technologies) were done as previously described [19 (link)]. LPS (#tlrl-peklps, InvivoGen) pre-incubation or post-treatment were specified in figure legends. Observation of cell morphology was performed with microscope. The detail information about cell culture, virus preparation and virus infection and luciferase assay referred to other reports [19 (link)].