THP-1 blue cells (InvivoGen; San Diego, CA) is a human monocytic leukemia cell line with an NFκB-inducible secreted embryonic alkaline phosphate (SEAP) reporter. The cells were cultured per the company’s protocol. They were first stimulated with flagellin at a range of concentrations (InvivoGen) (0–10 μg/mL) for 6 hours to determine the appropriate concentration. In the experiments where cells were exposed to VAs (0–2%), they were placed in an air-tight chamber and exposed to isoflurane or sevoflurane by a vaporizer using air as a carrier for 6 hours as we previously reported [20 (link)–22 ]. Intravenous anesthetic (propofol) (0–100 μΜ) was added to corresponding wells for 6 hours. NFκB activation was assessed by using the reaction buffer Quanti-Blue to quantitate SEAP in the medium per the company’s protocol. Samples were subjected to a spectrophotometer analysis at 655 nm. We also examined the effect of anesthetics using HEK-TLR5 NFκB reporter cells (InvivoGen) stimulated with flagellin (1 μg/mL) for 6 hours and HEK-TLR7 NFκB reporter cells (InvivoGen) stimulated with R837 (Imiquimod) (10 μg/mL) for 6 hours.
Thp1 blue cells
Manufactured by InvivoGen
Sourced in United States
THP1-Blue cells are a reporter cell line derived from the human monocytic THP-1 cell line. They constitutively express a secreted embryonic alkaline phosphatase (SEAP) reporter gene that is under the control of a NFkB-inducible promoter. The SEAP activity in the cell culture supernatant can be detected using a colorimetric or chemiluminescent assay, providing a simple and convenient method for monitoring NFkB activation in response to various stimuli.
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Lab products found in correlation
Monomac 6 cells, by Leibniz Institute DSMZ (3 mentions)
Streptomycin, by Leibniz Institute DSMZ (3 mentions)
Penicillin, by Leibniz Institute DSMZ (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Nf κb inducible reporter, by InvivoGen (2 mentions)
Endotoxin free certified fbs, by Thermo Fisher Scientific (2 mentions)
T 75 tissue culture flasks, by BD (2 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
Plasmotest kit, by InvivoGen (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Flagellin, by InvivoGen (1 mentions)
Quanti blue substrate, by InvivoGen (1 mentions)
Ox40l, by BioLegend (1 mentions)
Mouse fc block, by BD (1 mentions)
Anti mouse mhc 2, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Mercaptoethanol, by Merck Group (1 mentions)
Easysep biotin positive selection kit, by STEMCELL (1 mentions)
14 protocols using thp1 blue cells
1
Modulation of NFκB Signaling by Anesthetics
2
Cultivation of THP1-Blue and MonoMac-6 Cell Lines
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All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The THP1-Blue cells (InvivoGen, San Diego, CA, USA) were cultured in an RPMI 1640 medium (Mediatech Inc., Herndon, VA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 U/mL penicillin, 100 μg/mL phleomycin (Zeocin), and 10 μg/mL blasticidin S. Human monocyte-macrophage MonoMac-6 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were grown in an RPMI 1640 medium supplemented with 10% (v/v) FBS, 10 μg/mL bovine insulin, 100 μg/mL streptomycin, and 100 U/mL penicillin.
3
Dendritic Cell Activation and Signaling Analysis
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Immature bone marrow-derived dendritic cells (BMDCs) were derived as described in reference 24 (link). Cells were pulsed with Adjuplex at the concentrations shown in that study, washed, blocked in BD mouse Fc block (BD Biosciences), stained for surface markers with anti-mouse MHC-II (BioLegend), CD80 (Serotec), CD86 (BD Biosciences), CD11c (APC), CD40 (BioLegend), and OX40L (BioLegend), and subsequently acquired and analyzed using a FACSCalibur flow cytometer (BD Biosciences) and the FlowJo software (Tree Star). Thp1-Blue cells (InvivoGen) expressing TLR1/2, TLR2, TLR2/6, TLR4, TLR5, TLR8, NOD1, and NOD2 were stimulated with Adjuplex or PRR ligands (InvivoGen) for 24 h, and the supernatants were tested for secreted embryonic alkaline phosphatase (SEAP) expressed following NF-κB or AP1 activation, using QUANTI-Blue substrate (InvivoGen).
4
Isolating and Culturing Murine and Human Macrophages
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Macrophages were isolated from C57BL/6 mice (age 8–10 weeks) obtained from the Department of Experimental Biological Models of E.D. Goldberg Institute of Pharmacology and Regenerative Medicine. We performed this research according to EU Directive 2010/63/EU concerning the protection of animals used for scientific purposes, and it was approved by the Animal Care and Use Committee of the Goldberg Research Institute of Pharmacology and Regenerative Medicine, Tomsk NRMC (Protocol No. 171052020 from 05.18.20).
Macrophages were isolated from peritoneal exudate using an EasySep™Biotin Positive Selection Kit and Anti-Mouse F4/80 Antibody (both from StemCell Technologies, Vancouver, BC, Canada). The macrophages were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 10% (v/v) heat-inactivated, endotoxin-free fetal bovine serum (FBS) (Hyclone, GB), 20 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 50 mM mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 50 μg/mL gentamycin.
THP-1Blue cells obtained from InvivoGen (San Diego, CA, USA) [45 (link)] and human monocyte–macrophage MonoMac-6 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) [46 (link)] were cultured at 37 °C in a humidified atmosphere containing 5% CO2, as reported previously.
Macrophages were isolated from peritoneal exudate using an EasySep™Biotin Positive Selection Kit and Anti-Mouse F4/80 Antibody (both from StemCell Technologies, Vancouver, BC, Canada). The macrophages were cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 10% (v/v) heat-inactivated, endotoxin-free fetal bovine serum (FBS) (Hyclone, GB), 20 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 50 mM mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 50 μg/mL gentamycin.
THP-1Blue cells obtained from InvivoGen (San Diego, CA, USA) [45 (link)] and human monocyte–macrophage MonoMac-6 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) [46 (link)] were cultured at 37 °C in a humidified atmosphere containing 5% CO2, as reported previously.
5
THP1-Blue Cell NF-κB/AP-1 Reporter Assay
6
Culturing Human Monocytic Cell Lines
7
NF-κB Activation in Monocytic Cell Lines
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All cells were maintained in 5% CO2 at 37°C. THP-1 Blue cells (human monocytic leukemia line with an NF-κB–inducible reporter (Invivogen) and RAW264.7 cells carrying the NF-κB luciferase plasmid (IMGENEX, USA) were maintained in RPMI 1640 medium (Invitrogen) and Iscove's modified Dulbecco's medium (IMDM, Invitrogen), respectively. The media were supplemented with 10% heat-inactivated FBS (endotoxin-free Certified FBS; Invitrogen), 2% penicillin/streptomycin, 2 mM L-glutamine, and 10 mM HEPES (all from Gibco). The cells were seeded in T-75 tissue culture flasks (Falcon, USA) and used between passages 2 and 3.The THP-1 and RAW264.7 cell lines were extensively validated by the Invivogen and IMAGENEX respectively. For quality control, we have tested the cells for mycoplasma with PlasmoTest™ kit (Invivogen).
8
NF-κB Luciferase Assay in Monocytic Cell Lines
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THP-1 Blue cells (human monocytic leukemia line with an NF-κB-inducible reporter; Invivogen, United States) and RAW264.7 cells carrying the NF-κB luciferase plasmid (IMGENEX, United States) were maintained in RPMI 1640 medium (Life Technologies, United States) and Iscove’s modified Dulbecco’s medium (IMDM, Gibco), respectively. All media was supplemented with 10% heat-inactivated FBS/1% Antibiotic-Antimycotic/2 mM L -glutamine/10 mM HEPES (all from Gibco, United States). The cells were cultured in T-75 flasks (Falcon, United States) and used between passages 2 and 3. HDAC1 knock down and vector control stable cell lines were maintained in complete IMDM (Gibco) with puromycin (Bandyopadhaya et al., 2016b (link)). Cells were maintained in a humidified incubator at 37°C in 5% CO2.
9
Expression and Purification of TER119 scFv-SIINFEKL
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The TER119 scFv fused to SIINFEKL peptide antibody fragment was created as described elsewhere16 (link) and inserted in a pSectagA expression vector (Life Technologies) for expression in HEK293E cells under serum-free conditions with 3.75 mM valproic acid (Sigma–Aldrich) for 7 d. Proteins were purified from supernatant using immobilized metal ion affinity chromatography on an Akta FPLC system and by size-exclusion using a Superdex 75 column (GE Healthcare). After purification, purity was assessed by SDS/PAGE, concentration determined by nanodrop and endotoxin levels measured using THP-1 × Blue cells (InvivoGen). Final products were sterile-filtered and stored at −80 °C.
10
Cultivation of THP1Blue and MonoMac-6 Cells
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