THP-1 monocytes were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). THP-1 cells were cultured in RPMI 1640 cell culture medium containing 2 g/L glucose (Thermo Fisher Scientific, Vienna, Austria) and supplemented with 10% heat inactivated fetal calf serum and 45 units/mL (1%) penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C and 5% CO2 in a humidified atmosphere. The THP1-LuciaTM NF-κB cells used for NF-κB activity measurements were purchased from Invivogen (Toulouse, France). THP1-Lucia NF-κB cells were maintained in RPMI 1640 cell culture medium containing 4.5 g/L glucose and 2.383 g/L HEPES (Thermo Fisher Scientific) supplemented with 10% (v/v) heat inactivated FCS, 1% penicillin/streptomycin and 100 µg/mL of normocin (Invivogen, Toulouse, France) at 37 °C and 5% CO2 in a humidified atmosphere. To maintain the stability of the reporter gene cell clone, cells were grown under selection pressure with 100 µg/mL of zeocin (Invivogen, Toulouse, France) every second cell passage. Cell density was kept between 2∙105 and 1∙106 cells/mL and the number of cell passage did not exceed 20 passages.
Thp1 lucia nf κb cells
Manufactured by InvivoGen
Sourced in France
THP1-Lucia™ NF-κB cells are a stable human monocytic cell line that has been engineered to express the Lucia luciferase reporter gene under the control of the NF-κB promoter. This allows for the monitoring of NF-κB activation in response to various stimuli.
Automatically generated - may contain errors
Lab products found in correlation
Normocin, by InvivoGen (2 mentions)
Rpmi 1640 cell culture medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Zeocin, by InvivoGen (2 mentions)
Dual luciferase reporter dlr assay system, by Promega (2 mentions)
Raw246.7 cells, by American Type Culture Collection (2 mentions)
Pen strep, by InvivoGen (2 mentions)
Penicillin streptomycin, by InvivoGen (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Thp 1 monocytes, by Leibniz Institute DSMZ (1 mentions)
Coelenterazine substrate, by Nanolight Technology (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Cytation 3 imaging reader, by Agilent Technologies (1 mentions)
8 protocols using thp1 lucia nf κb cells
1
Culturing THP-1 Monocytes and NF-κB Reporter Cells
2
Infection of Mtb-H37Rv in THP1-Lucia Foam Cells
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THP1-LuciaTM NF-κB cells (Invivogen) were cultured in a comparable manner to wild type THP-1 cells to generate macrophages and foam cells. THP1-LuciaTM-derived foam cells were infected with Mtb-H37Rv for 4 h, then washed with PBS to remove extracellular bacteria. Cell culture supernatants were collected at 24 h post-infection and filtered twice through 0.22 μm centrifuge tube filters (Sigma). To measure luciferase activity, 10 μL of the supernatants were transferred to white-bottom 96-well plates and monitored for activity in the presence of 2 μg/mL of coelenterazine substrate (NanoLight Technology) with a Clariostar plate reader (BMG Biotech).
3
LILRB3 Signaling Activation Assay
4
LILRB3 Signaling Activation Assay
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THP-1-Lucia™ NF-κB cells were purchased from InvivoGen. Human anti-LILRB3 antibody with the N297A mutation was coated onto the plate to activate LILRB3 signaling, and plates coated with hIgG (N297A) were used as the control. The activation of NF-κB signaling was evaluated by monitoring luciferase signal. Infected THP-1 reporter cells were cultured for an additional month before stimulation with anti-LILRB3. For the NF-κB reporter assay conducted in 293T cells, an NF-κB-driven firefly luciferase reporter plasmid co-transfected with a plasmid encoding CMV-driven Renilla luciferase along with plasmids expressing LILRB3, TRAF2, or cFLIP were transfected into cells. The luciferase activity was detected using the Dual-Luciferase® Reporter (DLR™) Assay System (Promega). LILRB3 chimeric receptor reporter cells were constructed as we described68 (link)–70 (link), with LILRB3-ECD fused with the transmembrane and intracellular domains of paired immunoglobulin-like receptor β, which signals through the adaptor DAP-12 to activate the NFAT promoter.
5
Cell Culture Protocols for RAW246.7 and THP1-Lucia NF-κB Cells
6
Cell Culture Protocols for RAW246.7 and THP1-Lucia NF-κB Cells
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RAW246.7 cells were purchased from ATCC and cultured in DMEM supplemented 10% fetal bovine serum (FBS). THP1-Lucia™ NF-κB cells (NF-κB-inducible reporter monocytes) were purchased from InvivoGen (Cat# thp1-nfkb) and cultured in RPMI 1640, 2 mM l -glutamine, 25 mM HEPES, 10% heat-inactivated FBS, 100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 μg/ml).
7
NFkB Pathway Activation Monitoring
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Cultures, THP-1-Lucia NFκB cells, stably expressing an NFκB-inducible Lucia reporter construct, purchased from InvivoGen and the human embryonic kidney cell line HEK293-T (ATCC CRL-3216) were maintained at 37 °C in a humidified 5 % CO2 incubator. Both THP-1 cell lines were grown in RPMI-1640 supplemented with 10 % heat inactivated (HI, 30 min at 56 °C) fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin and split every 3-4 days (THP-1 cells: 4 x 10 6 cells/T75-flask in 20 ml, THP-1-LuciaNFκB cells: 10 x 10 6 cells/T75-flask in 20 ml). Medium of THP-1-Lucia NFκB was additionally supplemented with 100 µg/ml Normocin (InvivoGen) and every other passage with 100 µg/ml Zeocin. Activation of the NFκB-inducible Lucia reporter results in secretion of luciferase into the cell culture supernatant. HEK293-T cells were cultured in Dulbecco's Modified Eagle medium (DMEM) containing 10 % FBS, 100 U/ml penicillin and 100 µg/ml streptomycin.
8
Monitoring NF-κB Pathway Modulation
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To monitor the potential induction or suppression of the NF-κB signal transduction pathway by the test substances, the NF-κB reporter gene assay was performed with THP1-Lucia™ NF-κB cells following the provider's suggestion (InvivoGen 2017) with some modifications. Through expression of the integrated Luciferase reporter construct, modifications in the activation of the NF-κB pathway can be monitored. After sub-cultivating the cells, the remaining cell suspension was centrifuged (4 min, 200×g), the supernatant was discarded and the cells were resuspended in fresh medium. Subsequently, 100,000 cells/100 µL/well were seeded into a 96-well plate and incubated with the respective substances in triplicates for 20 h. After 2 h of incubation, cells were stimulated with 1 µL of a 1 µg/mL LPS solution to mimic inflammatory conditions. A solvent control incubated with LPS for 18 h served as the positive control. Additionally, another positive control containing 200 × 10 6 heat-killed Listeria monocytogenes (HKLM) per µL was measured. At the end of the incubation period, the plate was centrifuged (2 min, 200×g) and 10 µL of the supernatant was transferred into an opaque 96-well plate to determine luciferase activity. Luminescence was detected with the Cytation3 imaging reader (BioTek, USA) after the injection of 50 µL of QUANTI-Luc™.
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