Human monocytic THP1 cell line was obtained from American Type Culture Collection (ATCC), and was cultured in RPMI 1640 medium (Lonza, Verviers, belgium) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO USA), 2 mM L-glutamine (Lonza, Verviers, belgium), 1 mM sodium pyruvate (Lonza, Verviers, belgium), 0.05 mM 2-mercaptoethanol (Scharlau, Barcelona, Spain), 60 μg/ml gentamicin sulfate (Lonza, Verviers, belgium), 2.2 μg/ml amphotericin B solubilized (Sigma-Aldrich, St. Louis, MO USA). THP1-XBlueTM-MD2-CD14 and HEK-Blue™ TLR reporter cell lines were purchased from InvivoGen (InvivoGen, Toulouse, France) or home made63 (link)64 (link). THP1-XBlueTM-MD2-CD14 cells are derived from human monocytic THP-1 cell line, and stably express MD2 and CD14 to enhance signalling responses. Each of seven HEK293-Blue™ TLR cell lines was stably transfected with a specific human TLR gene (hTLR2, hTLR3, hTLR4, hTLR5, hTLR7, hTLR8, hTLR9). Both THP1-XBlueTM and HEK-Blue™ cell lines carry NF-κB- and AP-1- inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Once TLRs signalling is initiated, NF-κB and AP-1 will be activated, which will initiate the secretion of SEAP. Thus, SEAP activity in the cell supernatants can be used to quantify NF-κB activation. THP1-XBlueTM and HEK-BlueTM cells were cultured as previously described64 (link).
2 mercaptoethanol
Manufactured by Scharlab
Sourced in Belgium
2-mercaptoethanol is a clear, colorless liquid. It is a reducing agent commonly used in biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Lonza (3 mentions)
Sodium pyruvate, by Lonza (3 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Thp 1 cell line, by American Type Culture Collection (1 mentions)
Gentamicin sulfate, by Lonza (1 mentions)
Chloramphenicol, by Scharlab (1 mentions)
Peptone, by Solabia (1 mentions)
Kh2po4, by Scharlab (1 mentions)
Thp 1 monocytic cell line, by American Type Culture Collection (1 mentions)
Gentamycin sulfate, by Lonza (1 mentions)
Celastrol, by InvivoGen (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Rpmi medium, by Lonza (1 mentions)
4 protocols using 2 mercaptoethanol
1
Human Monocytic THP-1 Cell Culture and Reporter Lines
2
Analytical Techniques for Fungal Metabolites
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DON was from Romer Labs (Tulln, Austria). FB1 and FB2 were from Sigma (St. Louis, MO, USA), ortho-phthaldialdehyde was from Merck (Darmstadt, Germany) and 2-mercaptoethanol was from Scharlau (Sentmenat, Spain). Methanol HPLC grade, acetonitrile HPLC gradient grade and NaCl were from Fisher Scientific UK Limited (Loughborough, UK).
CGA was from Biokar (Barcelona, Spain). MGA was prepared in the laboratory according to Castellá et al. (1997) [69 (link)]. Peptone was from Biokar (Barcelona, Spain), KH2PO4 and chloramphenicol were from Scharlau (Sentmenat, Spain) and MgSO4·7 H2O was from Quality chemicals (Esparreguera, Spain). Malachite green (C48H50N4O4·2C2H2O4) was from Probus (Badalona, Spain) and agar was from Condalab (Torrejón de Ardoz, Spain).
CGA was from Biokar (Barcelona, Spain). MGA was prepared in the laboratory according to Castellá et al. (1997) [69 (link)]. Peptone was from Biokar (Barcelona, Spain), KH2PO4 and chloramphenicol were from Scharlau (Sentmenat, Spain) and MgSO4·7 H2O was from Quality chemicals (Esparreguera, Spain). Malachite green (C48H50N4O4·2C2H2O4) was from Probus (Badalona, Spain) and agar was from Condalab (Torrejón de Ardoz, Spain).
3
Cultivation of Human THP-1 Monocytes
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The human THP-1 monocytic cell line was obtained from ATCC (Manassas, USA). THP-1 cells were cultured in antibiotic-free RPMI 1640 medium (Lonza, Bornem, Belgium) with 10% FBS (Sigma–Aldrich, MO, USA), 2 mM L-glutamine (Lonza, Belgium), 1 mM sodium pyruvate (Lonza, Belgium) and 0.05 mM 2-mercaptoethanol (Scharlau, Barcelona, Spain). Cells were cultured at 37°C and 5% CO2.
4
Whey Hydrolysate Modulates NF-κB Cytokine Production
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In order to investigate the role of NF-κB in whey hydrolysate induced cytokine production, THP-1 monocytes were first differentiated into THP-1 macrophages as previously described [42 (link)]. First, THP-1 monocytes were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% decomplemented FBS, 2mM L-glutamine (Lonza, Verviers, Belgium), 1mM sodium pyruvate (Lonza, Verviers, Belgium), 0.05mM 2-mercaptoethanol (Scharlau, Barcelona, Spain), 60 μg/ml gentamycin sulfate (Lonza, Verviers, Belgium), and 2.2 μg/ml amphotericin B (Sigma Aldrich, Zwijndrecht, the Netherlands). Then, cells were seeded in a 24 wells plate at a concentration of 1x106 cells/well (in 0.5 ml medium). To differentiate the cells into macrophages 100 ng/ml PMA was added. After 48 hours, the PMA was removed and cells were washed twice with fresh medium. Cells were cultured for another 24 hours. Then, cells were incubated with either 10 μM of the NF-κB inhibitor celastrol (Invivogen, Toulouse, France) or medium (control) for 30 minutes. Next, 2 mg/ml intact whey protein or its hydrolysates were added. Cells were incubated for 24 hours, after which the supernatant was collected. TNFα and IL-10 levels were measured in the supernatant by ELISA according to the manufacturer’s protocol (eBioscience, San Diego, USA).
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