For TLR4 activation assay, 12 h prior to the transfections, HEK293T cells expressing humanTLR4, CD14 and MD2 (293/hTLR4A-MD2-CD14; Invivogen) were seeded into 12-well plates at a density of 0.5×106/well. Cells were then co-transfected with various amounts of HA-CLIP170 (50, 100, and 250 ng), pNF-κB-Luc (100 ng; Stratagene), and pRL-TK (50 ng; Promega) plasmids. The total amount of DNA was made constant by adding empty vector (pCMV-HA). Twenty-four hours post-transfection, cells were challenged with 300 ng of LPS for 12 h. Cells were then lysed and luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocols. Firefly luciferase activity was normalized to the Renilla luciferase activity and the reporter assays were expressed as mean fold induction over un-induced controls. For TLR9 activation assay, HEK293T cells were co-transfected with mTLR9 (100 ng) expression plasmid and other reporter plasmids mentioned above followed by induction with 1μg of Oligodeoxynucleotides (ODN) and measurement of luciferase activity. Assays were performed in triplicate, and the experiments were repeated thrice.
293 htlr4a md2 cd14
Manufactured by InvivoGen
Sourced in United States
The 293/hTLR4A-MD2-CD14 is a human embryonic kidney cell line that stably expresses the human Toll-like receptor 4 (TLR4) and its accessory proteins MD2 and CD14. This cell line is designed for the functional analysis of TLR4 signaling and screening of TLR4 agonists or antagonists.
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Lab products found in correlation
Penicillin, by Fujifilm (2 mentions)
Streptomycin, by Fujifilm (2 mentions)
Penicillin, by InvivoGen (2 mentions)
Streptomycin, by InvivoGen (2 mentions)
293 htlr4a md2 cd14, by Merck Group (2 mentions)
Hek293, by RIKEN BioResource Center (2 mentions)
Prl tk, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
293 htlr 4a, by InvivoGen (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Caco 2 cells, by Merck Group (1 mentions)
Mem non essential amino acids solution, by Fujifilm (1 mentions)
Caco 2 cells, by RIKEN BioResource Center (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Lx 2 cells, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by InvivoGen (1 mentions)
5 protocols using 293 htlr4a md2 cd14
1
TLR4 and TLR9 Activation Assays
2
Generation of TLR4-expressing HEK 293 Cells
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The pDUO-hTLR4A/MD2 (a pDUO plasmid co-expressing the human TLR4A and MD2 genes from InvivoGen) or pDUO-hTLR4A/CD14 plasmid (a pDUO plasmid co-expressing the human TLR4A and CD14 genes from InvivoGen (San Diego, CA, USA), were transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen/Life Technologies Corp., Grand Island, NY, USA). Two days later, cells were placed in selection medium (culture medium containing 10 µg/mL blasticidin). Clones were isolated from blasticidin-resistant cells by serial dilutions. Protein expression was screened by immunoblot analysis. A clone of 293-hTLR4A-MD2 and a clone of 293-hTLR4A-CD14 were selected for experiments. The other stably transfected HEK 293 cell lines: 293-Null, 293-hTLR4A, 293-hTLR4A-MD2-CD14, and 293-hMD2-CD14 were purchased from InvivoGen (San Diego, CA, USA).
3
Caco-2 and HEK293/TLR4 Cell Culture
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Caco-2 cells (human intestinal epithelial cells) were purchased from RIKEN BioResource Center (Tsukuba, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Moregate Biotech, Bulimba, Australia), 100 U/ml penicillin, and 100 μg/ml streptomycin (Wako Pure Chemical Industries); and 1% MEM non-essential amino acids solution (Wako Pure Chemical Industries), and was maintained at 37°C in a humidified atmosphere of 5% CO2. The medium was changed every other day. Caco-2 cells were activated by human recombinant tumor necrosis factor (TNF)-α (Merck Millipore, Burlington, MA, USA). Human embryonic kidney cells (HEK293, RIKEN BioResource Center) or their derivatives, which were stably transfected with the human toll-like receptor (TLR) 4a, MD2, and CD14 genes (293/hTLR4A-MD2-CD14; InvivoGen, San Diego, CA, USA), were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The 293/hTLR4A-MD2-CD14 cells were activated by lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA).
4
Assessing TLR4 Activity in HEK293 Cells
5
Immortalized HSC Cells Cultured
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LX-2 cells, immortalized human HSCs, were purchased from Merck Millipore (Billerica, MA, United States). In all experiments, the cells were subjected to no more than 15 cell passages. The cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, United States) containing 2% fetal bovine serum (Moregate Biotech, Bulimba, QLD, Australia), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Wako Pure Chemical Industries), and maintained at 37°C in a humidified atmosphere of 5% CO2. The medium was changed every other day. Human embryonic kidney cells (HEK293, provided by the RIKEN BioResource Center, Tsukuba, Japan) or their derivatives, which were stably transfected with the human Toll-like receptor (TLR) 4a, MD2, and CD14 genes (293/hTLR4A-MD2-CD14; InvivoGen, San Diego, CA, United States), were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. 293/hTLR4A-MD2-CD14 cells were activated by lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, United States) treatment.
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