For stimulation assays, 4 × 105 monocytes were exposed to ultrapure LPS (20 ng/ml) (catalog # tlrl-pelps, Invivogen) for either 2 or 24 hours. Alternatively, monocytes were exposed to IFNγ (catalog # 285-IF, R&D Systems) at a concentration of 20 ng/ml for 24 hours. The number of treated samples per individual depended on the total number of cells purified, and the priority on sample accrual was collation of IFNγ-treated followed by 24-hour LPS and then 2-hour LPS, respectively. Experiments were terminated simultaneously, ensuring identical incubation periods for all samples. A subset of samples was kept in the incubator without stimulation throughout this period to ascertain incubator effects. All experiments were completed within 48 hours of blood sample collection.
Tlrl pelps
Manufactured by InvivoGen
Sourced in United States
Tlrl-pelps is a synthetic ligand that activates the TLR7/8 receptor. It is a laboratory equipment product designed for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Tlrl stfla, by InvivoGen (2 mentions)
Flagellin, by InvivoGen (1 mentions)
Af 401 na, by R&D Systems (1 mentions)
Lipopolysaccharide lps, by InvivoGen (1 mentions)
Adi 905 173 100, by Enzo Life Sciences (1 mentions)
Ab54037, by Abcam (1 mentions)
A5316, by Merck Group (1 mentions)
Sc 514, by Santa Cruz Biotechnology (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Clenbuterol hydrochloride, by Merck Group (1 mentions)
4 protocols using tlrl pelps
1
Monocyte Stimulation Assay Protocol
2
Inflammasome Activation and Lipid Metabolism
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Lipopolysaccharide (LPS) (Escherichia coli; tlrl-pelps) and flagellin (Salmonella typhimurium; tlrl-stfla) were from Invivogen (San Diego, CA, USA). Adenosine triphosphate (ATP) (A2383), while nigericin (N7143), poly(dA:dT) (P0883), and PF-04620110 (PZ0207) were from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies were used: polyclonal rabbit anti-caspase-1 for mouse caspase-1 (1:1,000) (SC-514; Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal goat anti-IL-1β for mouse IL-1β (1:1,000) (AF-401-NA; R&D Systems, Minneapolis, MN, USA), polyclonal rabbit anti-ASC for mouse ASC (1:1,000) (ADI-905-173-100; Enzo Lifesciences, Farmingdale, NY, USA), polyclonal rabbit anti-DGAT1 for mouse DGAT1 (1:1,000) (ab54037; Abcam, Cambridge, MA, USA), and monoclonal mouse anti-β-actin (1:5,000) (A5316; Sigma-Aldrich).
3
Profiling Cytokine Responses to TLR Ligands
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Cells were seeded in 96-well plates and stimulated at 80% confluence with or without specific TLR ligands in 100 µl culture medium (triplicates); Pam3CysSerLys4 (P3CSK4; TLR2/1 ligand, 100 ng/ml, #L2000, EMCmicrocollection GmbH, Tübingen, Germany), Pam2CGDPKHPKSF (FSL-1; TLR2/6 ligand, 50 ng/ml, #L7000, EMCmicrocollection GmbH), polyinosinic:polycytidylic acid (poly (I:C); TLR3 ligand, 50 μg/ml, #27-4729-01, Amersham Pharmacia Biotech, Uppsala, Sweden), E. coli lipopolysaccharide (LPS) (TLR4 ligand, 100 ng/ml, #tlrl-pelps, InvivoGen, San Diego, CA), flagellin (TLR5 ligand, 1 μg/ml, #tlrl-stfla, InvivoGen), R848 (TLR7/TLR8 ligand, 1 μg/ml, #tlr-r848-5, InvivoGen), and CpG oligodeoxynucleotide (ODN) 2006 (TLR9 ligand, 20 μM, TIBMolBiol, Berlin, Germany). LPS was sonicated for 5 min prior to use. After 24 h supernatants were collected, centrifuged, and stored at -80 C°.
For quantification of cytokine responses, supernatants were thawed on ice and analyzed with a human 10-plex cytokine immunoassay (Bio-Rad) on a Bio-Plex 200 system (Bio-Rad) powered by Luminex xMAP Technology. The levels of IL-1β, IL-6, IL-8, IL-9, IL-10, IL-12 (p70), interferon-(IFN)-γ inducible protein (IP)-10, tumor necrosis factor (TNF)-α, IFN-γ, and vascular endothelial growth factor A (VEGF-A) were measured.
For quantification of cytokine responses, supernatants were thawed on ice and analyzed with a human 10-plex cytokine immunoassay (Bio-Rad) on a Bio-Plex 200 system (Bio-Rad) powered by Luminex xMAP Technology. The levels of IL-1β, IL-6, IL-8, IL-9, IL-10, IL-12 (p70), interferon-(IFN)-γ inducible protein (IP)-10, tumor necrosis factor (TNF)-α, IFN-γ, and vascular endothelial growth factor A (VEGF-A) were measured.
4
Functional Immune Response Assay
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Blood samples were collected between 9 and 10 A.M. after an overnight fast lasting at least 12 hours. Functional immune responses were assessed in vitro using a well-established LPS challenge protocol that reliably induces pro-inflammatory cytokine production [36] . Whole blood samples were cultured on RPMI Medium 1640 (1x) with L-Glutamine, Cat# 11875-093 (Invitrogen, Waltham, MA) for 6 hours at 37°C in a 5% CO2 tissue culture incubator under three conditions: in the presence of LPS alone, in the presence of LPS and the β₂-agonist CBL, and in the absence of both LPS and CBL (i.e. Control condition). Specifically, LPS-EB Ultrapure 5mg, 5 × 10 6 EU/ml, Cat# tlrl-pelps (InvivoGen, San Diego, CA) with working concentration of 0.1 μg/mL and Clenbuterol hydrochloride, Cat# C5423-10MG (MilliporeSigma, St. Louis, MO) with working concentration of 10 -6 M were used. After 6 hours of incubation, the cultured whole blood samples were centrifuged. Supernatant was collected and stored at -20°C.
Comprehensive details on sample collection, storage, dose determination of LPS and CBL, as well as experimental environment can be found in Supplementary Methods -SM1. Additionally, details of secondary analyses to determine if storage time affected immune protein levels within our sample were reported in Supplementary Methods -SM2.
Comprehensive details on sample collection, storage, dose determination of LPS and CBL, as well as experimental environment can be found in Supplementary Methods -SM1. Additionally, details of secondary analyses to determine if storage time affected immune protein levels within our sample were reported in Supplementary Methods -SM2.
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