Pharmacological reagents were obtained from the following manufacturers: Sigma (ML133, quinine, tetraethylammonium chloride (TEA), tetrapentylammonium (TPA), MCC950, lipopolysaccharide (LPS) from Escherichia coli O26:B6, and ATP), AdooQ (TRAM‐34 and PAP‐1), Alomone Labs (guangxitoxin‐1E), Tocris (dofetilide), Merck Millipore (Ac‐YVAD‐CMK), U.S Silica (Silica), Life Technologies (DNA [pEF/v5‐His A plasmid empty vector], Lipofectamine 3000), and Invivogen (ultrapure flagellin from Salmonella typhimurium and imiquimod). Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma). All other materials/reagents were obtained from Sigma unless otherwise stated.
Lipofectamine 3000
Manufactured by InvivoGen
Sourced in United States
Lipofectamine 3000 is a cationic lipid-based transfection reagent used for the delivery of nucleic acids into mammalian cells. It facilitates the uptake of plasmid DNA, siRNA, and other nucleic acids into the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Ml133, by Merck Group (1 mentions)
Epr16883, by Abcam (1 mentions)
G 20b 0014 c100, by Adipogen (1 mentions)
Ab209845, by Abcam (1 mentions)
Quinine, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Il 1β, by BD (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Quanti luc, by InvivoGen (1 mentions)
Tnf α, by BD (1 mentions)
Colchicine, by Merck Group (1 mentions)
Uric acid, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti gr 1, by Thermo Fisher Scientific (1 mentions)
Anti cd11b, by Thermo Fisher Scientific (1 mentions)
Crt0066101, by Bio-Techne (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
7 protocols using lipofectamine 3000
1
Inflammasome Activation Pathways
2
SEAP and Lucia Assays for cGAMP and DNA Sensing
3
Generation and Characterization of MSU Crystals
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MSU crystals were generated as described 12 (link). Briefly, we dissolved 1,68g of uric acid (Sigma-Aldrich) into 500mL of water containing 0,01M of NaOH by heating at 70°C, pH was adjusted to 7,1-7,2. Then, the uric acid solution was left at room temperature until the crystals formed under mild agitation. Crystals were then washed in ethanol, dried, weighted and thermally treated (250°C for an hour) and finally sonicated to obtain crystals <50µm in length. They were aliquoted in sterile PBS and frozen at -20°C until use. Poly-(dA:dT) and Lipofectamine 3000 were purchased from Invivogen and Invitrogen, respectively; LPS (Salmonella abortus equi), ATP and Colchicine from Sigma-Aldrich; ELISA kits (IL-1β, IL-6, TNFα, MPO) from Biotechne. Thioglycolate was home-made from brewer's thioglycolate (BD). The various antibodies used for Western blot were from Biotechne (anti-mouse IL-1β, AF401, and secondary HAF-109) or from Abcam (anti-tubulin) and Sigma-Aldrich (anti-vinculin). Anti-Gr1, Anti-CD11b, anti-CD14 and 7-AAD were obtained from eBioscience. CRT0066101 was provided from the lab of Romeo Ricci and is available from Tocris Bioscience; Anakinra was generously provided by Ommar S. Omarjee and Alexandre Belot (Lyon University, France) and Etanercept by Christian Von Frenckel (Liege University, Belgium).
4
RNA Interference and Immune Stimulation
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NHDF or tHF cells seeded in 12 well plates were transfected with 40uM siRNAs (SMAD3 and IRF7; Thermo Fisher Scientific) or miRNA mimics (custom designed; IDT) per well using Lipofectamine RNAiMax (Thermo Fisher Scientific) according to the manufacturer’s instructions. 33ug/mL ISD90 (IDT) and 16.5ug/mL polyI:C (Invivogen) was transfected into cells using Lipofectamine 3000 according to the manufacturer’s instructions.
5
Transfection and Mycoplasma Testing for HepG2 Cells
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Parental HepG2 cells (ATCC #85011430) and HepG2-derived KO cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich) at 37°C, 5% CO2 in a humidified incubator. Cells were routinely tested for mycoplasma contamination. DNA transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific) by preparing a DNA solution containing P3000 reagent (1.5 µl/µg DNA), and then forming complexes of DNA:Lipofectamine 3000 at a ratio of 2:3. Transfection with poly(I:C) (Invivogen #tlrl-pic; 1 µg/ml final) was performed using Lipofectamine 3000 as described above. For siRNA transfection, cells were transfected with 25 nM siRNA oligonucleotides using Lipofectamine RNAiMAX (ThermoFisher Scientific) according to the manufacturer's instructions.
6
Chloride Inhibitors Modulate 2,3-cGAMP Production
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The effect of chloride inhibitors on 2,3-cGAMP production was tested on human leukemia monocytic THP-1 cells. 1 × 106 THP-1 or RAW 264.7 cells were seeded in well plate. 1h later, cells were treated 100 μM NPPB, 150 μM DIDS, 200 μM IAA-94 overnight. Followed by transfection with 2×10−2 μg/μL HT-DNA via Lipofectamine 3000 reagent InvivoGen (San Diego, CA, USA) for 6 h. Cell pellets were lysed in M-PER™ Mammalian Protein Extraction Reagent (Thermo-Fisher Scientific, CA, USA) supplemented with cOmplete protease inhibitor cocktail (Roche). Lysed cells were centrifuged for 10 min at 14,000 g and 4 °C. Protein concentration of each lysate was measured using the Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, CA, USA). The 2′–3′-cGAMP content were measured by 2′–3′-cGAMP ELISA kit, Cayman Chemicals (Ann Arbor, MI, USA) according to the manufacturer’s instructions. Plates were read using SpectraMax™ i3/i3x Multi-Mode plate reader.
7
Poly(I:C)-Induced TLR3 Activation
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Cells were seeded (20,000/cm 2 ) for 24 hours, followed by antibiotics-free media replacement. A mixture of Lipofectamine 3000 (2.5 mL/mL; TFS) and poly(I:C) (2.5 mg/mL; InvivoGen) in OptiMEM (50 mL/mL; Gibco, TFS) was added to the medium. Cells were gently shaken and then incubated at 37 C. In case of inhibition of TLR3 stimulation, cells were pretreated for 1 hour at 37 C with bafilomycin A1 (100 nmol/L; InvivoGen), and then medium was replaced with fresh one and stimulation with poly(I:C) was performed as described above.
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