HepG2, HepG2.2.15, and Huh7 cells were cultured in DMEM cell culture media (Gibco) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin; HEK293T cells were cultured in DMEM cell culture fluid containing 10% FBS only. All cells were cultured at 37 °C and 5% CO2. The cells were transfected using Lipofectamine2000 (InvivoGen) according to manufacturer protocols (Lipofectamine 2000 2.5 µl with plasmids 1 µg). The supernatant of lentivirus isolated from HEK293T cells, which were co-transfected with sh-RAD51C, psPAX.2 and pMD2.G plasmids, and then HepG2 and Huh7 cells were infected at a ratio of culture fluid: virus liquid = 1:5. Polybrene (8 μg/ml) was added to increase the viral infection efficiency, and a RAD51C low-expressing cell line was established after puromycin (sellek, 2.5 μg/ml) selection.
Lipofectamine 2000
Manufactured by InvivoGen
Sourced in United States
Lipofectamine 2000 is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as plasmid DNA, siRNA, or mRNA, into eukaryotic cells. It facilitates the uptake of these molecules by the cells, enabling their expression or silencing. The product is widely used in research applications to study gene function and expression.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (15 mentions)
Poly 1 c, by InvivoGen (4 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Hek293t cell, by American Type Culture Collection (3 mentions)
Pam3csk4, by InvivoGen (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Lipopolysaccharides (lps), by InvivoGen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Nigericin, by Bio-Techne (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Poly 1 c hmw, by InvivoGen (2 mentions)
Propidium iodide, by ImmunoChemistry Technologies (1 mentions)
Mouse il 1β elisa kit, by BD (1 mentions)
Bb ci amidine, by Cayman Chemical (1 mentions)
Gsk484, by Cayman Chemical (1 mentions)
Goat anti rabbit igg af 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
45 protocols using lipofectamine 2000
1
Establishing RAD51C Knockdown Cell Lines
2
Investigating Inflammasome Regulation in Cells
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Cells were pre-treated with the following compounds where indicated prior to NLRP3 stimulation; MCC950 (15 nM, Cayman Chemical, Cambridge, UK) for 1 hr, YVAD (2 μg/ mL, Invivogen, San Diego, California) for 1 hr, OxPAPC (30 μg/ mL, Invivogen) for 1 hr, Amiloride (10 μM, 100 μM, Cayman Chemical, Cambridge, UK) for 1 hr, EIPA (10 μM, Cayman Chemical) for 1 hr, SPLUNC1-derived peptide, S18 (25 μM, gift from Spyryx Biosciences, Inc) for 4 hr or ouabain (100 nM, Cayman Chemical, Cambridge, UK) for 24 hr. Inflammasome stimulation was achieved using either LPS (10 ng/mL, Ultrapure EK, Invivogen) for 4 hr with the addition of ATP (5 mM, Invivogen, San Diego, California) for the final 30 min of stimulation, poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000, Invivogen, San Diego, California) for 1 hr, TcdB (10 ng/mL, Cayman Chemical, Cambridge, UK) for 1 hr or flagellin (10 ng/mL with Lipofectamine 2000, Invivogen, San Diego, California) for 1 hr for the final 1 hr of the LPS stimulation. ATP was dissolved in pre-warmed at 37°C medium (100 mM stock) and immediately (~2 min) added to the cells. All incubations were done in a humidified incubator at 37°C, 5% CO2. Supernatant, RNA and protein were collected and stored immediately following stimulation.
3
Silencing TREM-1 Modulates Inflammatory Responses
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The TREM-1 small interfering RNA (siRNA) (siTREM-1) and non-targeted scrambled control siRNA (siNTC) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Transient knockdown assays were performed using Lipofectamine 2000 (Invivogen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were normally seeded into 6-well culture plates 24 h prior to transfection. Once the cells had reached 60–80% confluence, the cells were transfected using Lipofectamine 2000 and with 20 pmol/ml siRNAs, as follows: control group, siNTC group, and siTREM-1 group. Knockdown efficiency was determined by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and western blotting. After RAW264.7 cells were transfected with siNTC or siTREM-1 for 48 h, cells were treated with 100 ng/ml LPS for 24 h in the presence of MH (50 µM) or vehicle; the control group was not transfected or treated with LPS. Afterwards, cells were collected to assess the corresponding indicators.
4
Inflammasome Activation Assay
5
FXR siRNA Knockdown in LO2 Cells
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The FXR siRNA (sense 5′-CAAGTGACCTCGACAACAA-3′) for human was synthesized by RiboBio Co., Ltd. (Guangzhou, China). LO2 cells were seeded onto 6-well plates and transfected with siRNA duplexes using Lipofectamine 2000 (Invivogen, San Diego, CA, United States) according to manufacturer’s instructions. The medium was replaced 6 h later, and then the cells were grown for an additional time up to 48 h. At the time point of 24 h before harvest, the siRNA-intervened cells were treated with emodin, UDCA or DXM.
6
Measuring IFIH-1 Mediated IFN-β Activation
7
Macrophage Immune Response Assay
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BMDMs, Raw264.7 macrophages and HEK293T cells were maintained in DMEM supplemented with 10% FBS and grown at 37 °C in 5% CO2 humidified incubator. BMDMs and Raw264.7 macrophages were transfected with poly(dA:dT) (1 μg/ml), poly(dC:dG) (1 μg/ml), dsVACV70mer (1 μg/ml) or HSV60mer (1 μg/ml) using either Lipofectamine® 2000 according to the manufacturer's instruction or treated with Pam3CSK4 (10 ng/ml; InvivoGen), poly(I:C) (200 ng/ml; InvivoGen) or LPS (Serotype O111:B4; 1 μg/ml; InvivoGen) for 6 h or 18 h. BMDMs were also transfected with various serotypes of LPS (O111:B4, O55:B5, E. coli K12, P. gingivalis, S. Minnesota; InvivoGen and O127:B8; Sigma-Aldrich; 2 μg/ml) for 18 h using FuGENE® HD (Promega) according to the manufacturer's instruction. The cells and culture media were immediately used for RNA isolation and enzyme-linked immunosorbent assay (ELISA) experiments.
8
Poly I:C-induced Signaling in HEK293 Cells
9
Knockdown of ILF3 and EIF2AK2 in HeLa and A549 cells
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HeLa and A549 cell lines were maintained in DMEM supplemented with 10% (v/v) foetal calf serum and 1% Penicillin/streptomycin at 37°C and 5% CO2. Transfections of poly(I:C) (2 μg/ml, HMW, tlrl-pic; Invivogen) were performed using Lipofectamine 2000 according to the manufacturer's instructions. Four hours post-transfection, cells were collected for downstream applications. For IFN-β stimulation, HeLa cells were treated with 100U of recombinant human IFN-β (Peprotech, 300–02BC) for 4 h and harvested. Knock-down experiments were performed by two consecutive rounds of transfection with siRNA pools against ILF3 (L-012442–00-0005, Dharmacon) or EIF2AK2 (L-003527–00-0005, Dharmacon) in HeLa or A549s. As a negative control, a non-targeting siRNA pool was used (D-001810–10-05, Dharmacon). Individual siRNAs against both major ILF3 isoforms were adapted from Guan et al., (21 (link)). For NF110 (Sense-CUACGAGAGCAAAUUCAA C[dT][dT], antisense–GUUGAAUUUGCUCUCGUAG[dT][dT]), and NF90 (sense-G[mC]CCACC[mU]UUG[2flC]UU[2flU]UUAU[dT][dT], antisense- AUAA[mA]AAGCAAAGGUGG[2flG]C) siRNAs were used. Briefly, cells were seeded at 50–60% confluency and transfected with 25 nM siRNAs using Dharmafect. After 24 h, cells were split and transfected with a second round of siRNAs. Cells were collected for downstream processing 72 h after the first transfection.
10
Virus Sensing and IFN-β Luciferase Assay
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PK-15 or IPEC-J2 cells were grown in 24-well plates. In selected experiments, the recombination or empty expression plasmids were cotransfected with the pGL3-pIFN-β-Luc and pRL-TK (an internal control for the normalization of the transfection efficiency) using Lipofectamine 2000 (InvivoGen, USA). After transfection for 24 h, poly(I:C) or VSV-GFP virus were transfected or infected for 16 h. The cells were then lysed, and the firefly luciferase and Renilla luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega, USA). Data were shown as the relative firefly luciferase activities normalized to the Renilla luciferase activities from three independently conducted experiments.
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