The TLR7 agonist SZU-101 was synthesised as described in Additional file 2 : Figure S2. HEK-BLUE hTLR7 cells were purchased from InvivoGen. The cells stably expressed human TLR7 and a SEAP reporter, which can be used to detect TLR7 agonism through the activation of NF-kB signalling. The cells were maintained in selective DMEM growth medium with an additional 10 μg/ml blasticidin and 100 μg/ml Zeocin™. After incubation with different doses of SZU-101, the cells were tested using the HEK-BLUE detection kit according to the manufacturer’s instructions. The TLR7 agonist imiquimod and purified mouse TNF-α were used as positive controls. The induction of TLR7 activation can be visualised and assessed by reading the OD at 620–655 nm.
Hek blue htlr7 cells
Manufactured by InvivoGen
Sourced in United States
HEK-BLUE hTLR7 cells are a stable reporter cell line derived from HEK293 cells and engineered to express the human Toll-like receptor 7 (hTLR7). These cells are designed to monitor the activation of hTLR7 in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Zeocin, by Thermo Fisher Scientific (1 mentions)
Heracell 150i, by Thermo Fisher Scientific (1 mentions)
Hek blue hnod2, by InvivoGen (1 mentions)
Hela cells, by InvivoGen (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Polyinosinic polycytidylic acid poly 1 c, by Merck Group (1 mentions)
Reparixin, by MedChemExpress (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Emax precision microplate reader, by Molecular Devices (1 mentions)
Quanti blue solution, by InvivoGen (1 mentions)
Nih 3t3 cell line, by American Type Culture Collection (1 mentions)
Hep 2 cells, by American Type Culture Collection (1 mentions)
C57bl 6j, by Charles River Laboratories (1 mentions)
C57bl 6 mice, by American Type Culture Collection (1 mentions)
Wehi231, by American Type Culture Collection (1 mentions)
7 protocols using hek blue htlr7 cells
1
Evaluation of TLR7 Agonist SZU-101 Activity
2
TLR7 Agonism Assay with SZU-106
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SZU-106 was synthesized as described above. HEK-BLUE hTLR7 cells were purchased from InvivoGen (San Diego, CA). This cell line stably expresses human TLR7 and a SEAP reporter, which can be used to detect TLR7 agonism through the activation of NF-kB signaling. The cells were maintained in selective DMEM growth medium with an additional 10 μg/ml blasticidin and 100 μg/ml Zeocin™ (Fisher Scientific, #R25001, USA). After incubation with different doses of SZU-106 for 2 days, the reporter gene SEAP expression was detected using the HEKBLUE detection kit according to the 'manufacturer's instruction. The TLR7 agonist R848 was used as the positive control.
3
Cell Culture Protocols for Diverse Cell Lines
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HEK-Blue™ hNOD2 and HEK-Blue™ hTLR7 cells were obtained from Invivogen and cultured in DMEM GlutaMAXTM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 30 µg/mL blasticidin, and 100 µg/mL zeocin. HeLa cells (human epithelial cell line from adenocarcinoma) were obtained from Invivogen and cultured in DMEM GlutaMAXTM supplemented with 10% FBS and 1% (v/v) penicillin/ streptomycin. Immortalized Ho-1u-1 cells (a human cell line from the floor of mouth squamous cell carcinoma) were obtained from GIMAP (St Etienne, France) and propagated in DMEM/Ham’s F12 nutrient mixture (1:1) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. Immortalized DC2.4 cells (a murine bone-marrow-derived dendritic cell line) were obtained from InvivoGen (Toulouse, France) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 10 mM Hepes, and 50 μM β-mercaptoethanol. All cell lines were maintained in a 37 °C incubator (Heracell 150i, Thermo Scientific) under 5% CO2 and 95% humidity.
4
Culturing Mouse Melanoma and TLR7 Cells
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Mouse B16-F10 melanoma cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China) and cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. HEK-BLUE hTLR7 cells were purchased from InvivoGen (San Diego, CA, USA) and maintained in selective DMEM supplemented with 10 μg/mL blasticidin (Thermo Fisher Scientific, USA) and 100 μg/mLZeocin (San Diego, CA, USA). 5-Aza was purchased from Apexbio Technology LLC (Houston, TX, USA), polyinosinic–polycytidylic acid (poly: IC) was purchased from Sigma (St. Louis, MO, USA), and reparixin was obtained from MedChem Express LLC (Princeton, NJ, USA).
5
TLR Activation Assay in HEK Cells
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Cells were seeded in 96-wells plates (HEK-Blue hTLR7 Cells (InvivoGen): 70,000 cells per well; 293-mTLR9-luc cells70 (link) 40,000 cells per well) and incubated for 24 h. Then they were stimulated in triplicates for 18 h with the supernatant of the stimulated B cells (C57BL/6 (n = 3)), Tlr3−/− Tlr7−/− Tlr9−/− (n = 3), EGT-315 B6 (n = 1) and EGT-315 Tlr3−/− Tlr7−/− Tlr9−/− (n = 1), with medium as a negative control and with R848 (1 μg/ml), and 1668 PTO-ODN (1μM, Phosphorothioate oligo-deoxynucleotide), respectively as a positive control. 20 µl supernatant of the stimulated HEK-Blue hTLR7 Cells were added to 180 µL QUANTI-Blue Solution (InvivoGen) per well. After 1 h incubation at 37 °C the optical density (OD) at 650 nm was measured using an Emax precision microplate reader (Molecular Devices). The HEK mTlr9 cells were lysed by adding 50 µl 1 x Reporter Lysis Buffer (Promega) and freezing (-80 °C) and thawing them (RT) four times. The firefly luciferase activity was measured using Berthold Detection Systems (Pforzheim, Germany).
6
Characterization of Immune Cell Responses in Transgenic Mice
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Mice were kept under Specific pathogen free (SPF) conditions in IVC cages with a 12 h/12 h light dark cycle, a humidity of 55% + /- 5% at a room temperature of 21 + /− 1 °C. The following mouse strains were used in this study: C57BL/6 J from Charles River (Germany); hA3 transgenic mice26 (link), which contain the human Apobec3 gene locus, were crossed to generate EGT-315 Tlr3−/−Tlr7−/−Tlr9−/− hA3 mice; Tlr767 (link)-and Tlr968 (link)-deficient mice were used to isolate primary B cells for in vitro activation. Tlr3−/−Tlr7−/−Tlr9−/− triple deficient11 (link) mice; and T-bet deficient C57BL/6 mice (Tbx21 deficient69 (link)). For in vitro infections with ERV-GFP we used the B cell lymphoma WEHI-231 (ATCC CRL-1702), NIH-3T3 cell line (ATCC CRL-1658) and primary mouse embryonic fibroblasts (MEF) isolated from C57BL/6 mice. HEK-Blue hTLR7 Cells (InvivoGen) and HEK mTLR9 cells were used as reporter cells for Tlr7 and Tlr9 ligands respectively70 (link). For the indirect immunofluorescence assay HEp-2 cells (ATCC CCL-23) were used. For in vitro B cell activation we used 40LB cells37 (link).
7
Evaluating TLR7 Agonism Activity
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HEK-BLUE hTLR7 cells were purchased from InvivoGen (San Diego, CA, USA). The cells stably expressed human TLR7 and a SEAP reporter, which can be used to detect TLR7 agonism through the activation of NF-kB signaling. The cells were maintained in selective DMEM supplemented with 10 μg/mL blasticidin and 100 μg/mL Zeocin™. After incubation with different doses of SZU-106, the cells were tested using a HEK-BLUE detection kit, according to the manufacturer’s instructions. The TLR7 agonist imiquimod and SZU-101 were used as positive controls. The experiments were repeated 3 times. The induction of TLR7 activation was visualized and assessed by measuring the OD value at 620–655 nm.
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