THP-1 cells were seeded in 96-well flat-bottom tissue culture plates at 1 × 105 cells/well in 200 µL. Cells were primed with E. coli LPS (100 ng/mL), P. gingivalis LPS (100 ng/mL), T. denticola LPS (100 ng/mL), T. forsythia LPS (100 ng/mL), P. gingivalis OMVs (100 ng protein/mL), T. denticola OMVs (100 ng protein/mL), T. forsythia OMVs (100 ng protein/mL), or left unprimed for 4 h at 37°C in a 5% CO2 incubator. Cells were then stimulated with positive inflammasome activation controls Nigericin (10 µM) (Invivogen, USA), Silica (125 mg/mL) (US Silica, USA), or transfected with Poly(dAdT) (250 ng/mL) (Invitrogen, USA) using lipofectamine LTX (Invivogen, USA) as per the manufacturer’s instructions for a further 6 h. Primed cells were also treated with P. gingivalis, T. denticola, and T. forsythia OMVs at OMV: cell ratios of 10:1, 50:1, and 100:1 for 6 h or E. coli, P. gingivalis, T. denticola, and T. forsythia LPS (100 µg/mL) for 6 h. Cellular supernatants were collected and analyzed for IL-1β secretion by a human IL-1β ELISA Kit (Jomar Life Research, VIC, Australia) according to the manufacturer’s instructions.
Poly da dt
Manufactured by Thermo Fisher Scientific
Sourced in United States
Poly(dA:dT) is a synthetic double-stranded DNA polymer composed of alternating deoxyadenylic acid (dA) and deoxythymidylic acid (dT) residues. It is commonly used as a model system for the study of DNA structure and function in various applications within life science research.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (2 mentions)
Poly 1 c, by Merck Group (2 mentions)
Cgamp, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ifn β, by R&D Systems (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Nigericin, by InvivoGen (1 mentions)
Flagellin, by Thermo Fisher Scientific (1 mentions)
Z vad, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Poly da dt, by Merck Group (1 mentions)
Si control, by GenePharma (1 mentions)
Poly 1 c, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
5 ppprna, by InvivoGen (1 mentions)
Cgamp, by InvivoGen (1 mentions)
9 protocols using poly da dt
1
Inflammasome Activation in THP-1 Cells
2
NLRP3, NLRC4, and AIM2 Inflammasome Activation
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To trigger conventional NLRP3 inflammasome activation, J774A.1 cells were primed with 0.25 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) for 3 h and then treated with 2.5 mM ATP (Sigma-Aldrich) for 30 min with or without 10 µM Y-VAD (InvitroGen, Carlsbad, CA, USA), a selective caspase-1 inhibitor. To induce NLRC4 or AIM2 inflammasome, J774A.1 cells were primed as described above and then transfected with 0.5 μg/ml flagellin (InvitroGen) or 1 μg/ml poly(dA:dT) (InvitroGen) for 3 h, using Lipofectamine 2000 (InvitroGen) according to the manufacturer's protocol.
Flag-tagged caspase-11 (Flag-Caspase-11) and either Flag-tagged wild-type GSDMD (Flag-GSDMD [WT]) or D276A mutant GSDMD (Flag-GSDMD [D276A]) were transfected into HEK293T cells. Flag-GSDMD (D276A) was constructed in our laboratory. Then, HEK293T cells were treated with 20 μg/ml LPS for 4 h with or without 10 µM Z-VAD (InvitroGen), a pan-caspase inhibitor. Alternatively, HEK293T cells were transfected with Flag-tagged wild-type C-terminal domain of GSDMD (Flag-GSDMD-CT [WT]), wild-type N-terminal domain of GSDMD (Flag-GSDMD-NT [WT]), or 4A mutant N-terminal domain of GSDMD (Flag-GSDMD-NT [4A]) and cultured for 48 h.
Flag-tagged caspase-11 (Flag-Caspase-11) and either Flag-tagged wild-type GSDMD (Flag-GSDMD [WT]) or D276A mutant GSDMD (Flag-GSDMD [D276A]) were transfected into HEK293T cells. Flag-GSDMD (D276A) was constructed in our laboratory. Then, HEK293T cells were treated with 20 μg/ml LPS for 4 h with or without 10 µM Z-VAD (InvitroGen), a pan-caspase inhibitor. Alternatively, HEK293T cells were transfected with Flag-tagged wild-type C-terminal domain of GSDMD (Flag-GSDMD-CT [WT]), wild-type N-terminal domain of GSDMD (Flag-GSDMD-NT [WT]), or 4A mutant N-terminal domain of GSDMD (Flag-GSDMD-NT [4A]) and cultured for 48 h.
3
siRNA Knockdown of TRIM56 in Myeloma Cells
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Small interfering RNAs (siRNAs) against TRIM56 (T56si-1 and T56si-2) and negative control (si-control) were synthesized from GenePharma (Shanghai, China). Poly (I:C) (a dsRNA surrogate) and poly (dA:dT) (a dsDNA surrogate) were purchased from Sigma (Sigma, St. Louis, MO, USA). Transfection with sicontrol, T56si-1, T56si-2, poly (dA:dT), or Poly (I:C) into RPMI8226 or U266 cells was performed using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer's instructions.
4
Viral Infection and Immune Stimulation Assay
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HSV-1 was a gift from S. Paludan (Aarhus University, Denmark). MVA was a gift from I. Drexler (Düsseldorf University, Germany). SeV Cantell strain was from ECACC. Serial dilutions of SeV indicated the optimal dilution to elicit IFN without causing cell death as 1:200, which was used in all experiments. All other viruses were used at a multiplicity of infection of 10. Cell stimulants used were 70-mer dsDNA (70-mer) (9 (link)); poly(dA-dT), poly(I:C) (both Sigma-Aldrich); cGAMP and 5′pppRNA (both InvivoGen). 70-mer, poly(dA-dT), poly(I:C), 5′pppRNA, and cGAMP were delivered to cells by transfection using Lipofectamine®2000 (Invitrogen), at the concentrations indicated in figure legends. IL-1α (PBL Interferon Source) and TNFα (PeproTech) were also used.
5
Molecular Mechanisms of Innate Immunity
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Anti-TBK1/NAK (3504), anti-phospho-TBK1/NAK (5483), anti-IRF3 (10949), anti-phospho-IRF3 (4947), anti-NF-κB-p65 (8242), anti-phospho-NF-κB-p65 (3033), anti-TRAF6 (8028), anti-ub (20326), anti-ub-k63 (5621), anti-Myc-Tag (2276) and anti-Flag-Tag (14793) were purchased from Cell Signaling Technology (USA). Anti-phospho-IRF-3(Ser396) (SAB5700435), anti-HA-tag (H6908), anti-β-actin (A5441), and anti-Flag M2 Affinity Gel (A2220) were purchased from Sigma (USA). Anti-ub-k48 (EP8589) was purchased from Abcam (UK). Protein G Sepharose 4 Fast Flow was from GE Healthcare (Sweden). HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies for Western blot were purchased from Santa Cruz (USA). JetPRIME kit was obtained from Polyplus Transfection (France). Anti-pA151R polyclonal antibody was produced in rabbit by immunization with recombinant pA151R. Double-Luciferase Reporter Assay Kit was from Promaga (USA). 2’3’-cGAMP, poly(dA:dT), and Lipofectamine 3000 were purchased from Invitrogen (USA). 3-Methyladenine (3-MA, HY-19312), MG-132 (HY-13259), E-64 (HY-15282), and Z-VAD-FMK (HY-16658B) were from MedChemExpress (MCE) (USA).
6
Induction of Inflammasome Activation in Human Salivary Gland Cells
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Human salivary gland epithelial cell line (HSG) was maintained in DMEM (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Thermo Fisher Scientific). HSG cells were stimulated with 1,000 U/ml recombinant IFN-α (R&D Systems, Minneapolis, MN, USA) or 1,000 U/ml IFN-β (R&D Systems) in DMEM containing 10% FBS. For AIM2 inflammasome activation, HSG cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) reagents in Opti-MEM media (Thermo Fisher Scientific). After washing with Opti-MEM media, cells were stimulated with poly(dA:dT) 2 µg/ml (Invitrogen) for 6 h to 2 days in Opti-MEM media. For NLRP3 inflammasome, HSG cells were stimulated with Nigericin 10 µM (Sigma-Aldrich, St. Louis, MO, USA) for 12 h to 2 days in DMEM media (Thermo Fisher Scientific). Culture supernatants were collected, and cells were used for western blot or flow cytometry analysis. Caspase-1 inhibitors, VX765 (InvivoGen, San Diego, CA, USA), Caspase-3 inhibitor, Z-DEVD-FMK (R&D Systems), and Caspase-8 inhibitor, Z-IETD-FMK (R&D Systems) were pretreated for 1 h before poly(dA:dT) and Nigericin stimulation.
7
Transcriptomic Analysis of innate immune responses
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ncgRNA and PAF1 KO cells were plated at a density of 6.0 × 105 cells per well in 6-well plates and incubated at 37 °C for 24 hours. Cells were then stimulated with poly I:C (2.0 μg/mL, Tocris), poly dA:dT (1.0 μg/mL, InvivoGen), LPS (1.0 μg/mL, eBioscience), IFN-β (50 U/mL, R&D Systems), and TNF-α (100 ng/mL, Gibco Thermofisher) at the given concentrations. For poly I:C and poly dA:dT, transfection was performed with Lipofectamine 2000 (ThermoFisher) per manufacturer instruction. After 3 hours post-stimulation, all samples were collected with Trizol reagent (Ambion) and stored at − 80 °C. Total cellular RNA was extracted and purified with the RNeasy mini kit (Qiagen). At the sequencing facility, RNA samples were prepared with the QuantSeq3’ mRNA-Seq Library kit for Illumina (Lexogen GmbH), following a standard protocol [84 (link)]. The resulting cDNA libraries were sequenced on an Illumina NextSeq500 system with approximately 4 million reads per sample.
8
Biotinylated HSV-60 Probe Binding Assay
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In total, 60 bp HSV-60 was biotinylated with a biotin 3′-end DNA labeling kit as a DNA probe. The DNA-binding reaction was carried out with the LightShift Chemiluminescent EMSA kit (Thermo Scientific) according to the manufacturer’s instructions. A recombinant mouse RPSA-DNA complex was identified by electrophoresis on a 4% polyacrylamide gel. For the competition, we used 60 bp HSV-60, 70 bp VACV-70 (Invivogen), poly dA:dT, and poly dI:dC (Thermo) unlabeled probes.
9
Mucoepidermoid Carcinoma Cell Stimulation
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The human pulmonary mucoepidermoid carcinoma cell line NCI-H292 (ATCC CRL-1848) was maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37C in a humidified atmosphere with 5% (v/v) CO2. The NCI-H292 cells were expanded in 24-well plates and maintained in serum-free RPMI 1640 medium for 6 h before stimulation with mithramycin A (Cayman Chemical, Ann Arbor, MI, USA) for 30 min or with ML-60218 (Cayman Chemical) for 2 h. Subsequently, the cells were transfected with 0.5 µg of poly(dA:dT; Thermo Fisher Scientific) using polyethylenimine MAX (Polysciences, Warrington, FL, USA) and incubated for 6 h. The transfected cells were subsequently exposed to 25 µg/mL poly(I: C; Sigma-Aldrich, St. Louis, MO, USA) and 4 ng/mL TGF-α (R&D Systems, Minneapolis, MN, USA) for 6 h at 37C. For plasmid stimulation, the cells were transfected with 0.5 µg of pcDNA6.2-EGFP in 24-well plates, using FuGENE HD (Promega, Madison, WI, USA). For cGAMP (Thermo Fisher Scientific)-mediated cellular stimulation, cells were incubated in the presence of cGAMP for 16 h.
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