TC-1 cells were cultured in Dulbecco minimal essential medium (DMEM; Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClone), 1 mM HEPES buffer (Life Technologies), and 0.1 mM nonessential amino acids (Life Technologies) (complete DMEM; cDMEM), and plated at 2 x 105 viable cells per well in 24-well plates. After overnight culture, TC-1 cells were infected with F. tularensis LVS or SchuS4 at a multiplicity of infection (MOI) of 1:1 (bacterium-to-TC-1 cell ratio) for 2 hours, washed with PBS, incubated with 50 μg/ml gentamicin for 45 minutes, washed twice with warm PBS, and cDMEM with or without recombinant cytokines was added to the wells, as indicated. All cytokines (IFN-γ, TNF, IL-17A, and IL-22) were obtained from BioLegend, and used in experiments at a concentration of 100 ng/ml, with the exception of TNF, which was used at a concentration of 50 ng/ml. Cultures were incubated at 37°C in 5% CO2 for the remainder of the experiment. Bacterial uptake or recovery was determined after initial infection, and after 72 hours of culture, by washing the monolayers once with warm PBS, followed by lysis of infected cells with sterile distilled water, plating on agar, and counting. Supernatants were obtained from the cultures after 72 hours for nitrite analyses.
Tumor necrosis factor (tnf)
Manufactured by BioLegend
Sourced in United States, United Kingdom
TNF is a cytokine that plays a central role in inflammation and the immune response. It is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by BioLegend (10 mentions)
Ifn γ, by BD (6 mentions)
Il 10, by BioLegend (5 mentions)
Il 12p40, by BD (4 mentions)
Interleukin 6 (il 6), by BD (4 mentions)
Il 10, by Thermo Fisher Scientific (4 mentions)
Il 1β, by BioLegend (3 mentions)
Il 17, by BioLegend (2 mentions)
Ifn β, by BioLegend (2 mentions)
Il 1α, by R&D Systems (2 mentions)
Interleukin 2 (il 2), by BD (2 mentions)
Il 1α, by BioLegend (2 mentions)
Interleukin 4 (il 4), by BioLegend (2 mentions)
Ifn γ, by BioLegend (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Il 10, by BD (2 mentions)
Fix perm intracellular staining kit, by BD (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Brefeldin a, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
31 protocols using tumor necrosis factor (tnf)
1
Cytokine Modulation of Francisella tularensis Infection
2
Flow Cytometric Analysis of Inflammatory Cells
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Flow cytometry was performed essentially as previously described [16 (link),19 (link)] using FACSVerse (BD Biosciences) and data analyzed using the FACSuite software. TNF+ microglia (CD11b+CD45dim), TNF+ macrophages (CD11b+CD45highGr1−) and TNF+ granulocytes (CD11b+CD45highGr1+) were identified as previously detailed [16 (link),19 (link)]. Control mice and mice allowed to survive for six and 24 hours after pMCAO were treated intravenously with either saline, XPro1595 or etanercept 30 minutes after surgery.
Prior to fixation, cells were stained for live/dead cells for 30 minutes at 4°C using a Fixable Viability Dye eFluoro 506 (eBioscience, Hatfield, UK) diluted in PBS. A total of 1,000,000 events were collected using forward scatter (FSC) and side scatter (SSC) and analysis of the live/dead gate revealed comparable numbers of dead cells in all the samples. In addition, blood and spleen samples were collected and analyzed for CD45, CD11b, Gr1, and CD3 expression.
Positive staining for TNF (Biolegend, Copenhagen, Denmark), CD11b, CD45, Gr1 and CD3 (BD Pharmingen, Albertslund, Denmark) was determined based on fluorescence levels of the respective isotype controls (Biolegend and BD Pharmingen). The mean fluorescence intensity (MFI) was calculated as the geometric mean of each population in the TNF, CD45 and CD11b positive gates, respectively.
Prior to fixation, cells were stained for live/dead cells for 30 minutes at 4°C using a Fixable Viability Dye eFluoro 506 (eBioscience, Hatfield, UK) diluted in PBS. A total of 1,000,000 events were collected using forward scatter (FSC) and side scatter (SSC) and analysis of the live/dead gate revealed comparable numbers of dead cells in all the samples. In addition, blood and spleen samples were collected and analyzed for CD45, CD11b, Gr1, and CD3 expression.
Positive staining for TNF (Biolegend, Copenhagen, Denmark), CD11b, CD45, Gr1 and CD3 (BD Pharmingen, Albertslund, Denmark) was determined based on fluorescence levels of the respective isotype controls (Biolegend and BD Pharmingen). The mean fluorescence intensity (MFI) was calculated as the geometric mean of each population in the TNF, CD45 and CD11b positive gates, respectively.
3
Immunoblot Analysis of Apoptosis Markers
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Cell lysates were harvested from cells infected in TC-treated plates with lysis buffer and run on 4–12% NuPAGE gels (Invitrogen). Proteins were transferred to PVDF membrane (Millipore) and blotted with rat anti–mouse caspase-8 (1G12; Enzo Life Sciences), rabbit anti–caspase-3 (9662; Cell Signaling) and β-actin (Sigma), followed by HRP-conjugated secondary antibodies. Cytokine release was measured by ELISA on cell supernatants using capture and detection antibodies against TNF (BioLegend), IL-6 (BD), and IL-12p40 (BD).
4
Quantification of NMV Internalization in HCAEC
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In order to quantify NMV internalisation, HCAEC (5 × 104) were seeded onto 24-well tissue culture plates and cultured overnight. The next day HCAEC were incubated with media alone, media + TNF (1 ng mL−1; R&D Systems, Abingdon, UK), media + anti-ICAM-1 (100 ng mL−1; Biolegend, London, UK, catalogue number: 322704) or media + TNF + anti-ICAM-1 for 4 h. Consequently fluorescently labelled NMVs (0.4 × 103 µL−1) were added and cells incubated for 2 h at 4 °C, room temperature or 37 °C (as specified in the corresponding figure legends). The use of low temperatures to inhibit endocytosis has been used by others to demonstrate internalisation is a metabolically active process i.e. endocytosis dependent59 (link),60 (link). Cells were then washed to remove excess NMVs and detached using a trypsin/EDTA solution. Cells were washed and, immediately prior to flow cytometry analysis, Trypan blue (1 mg mL−1) was added to each sample in order quench fluorescence of residual surface bound NMVs61 (link),62 (link), thus ensuring any fluorescent signal detected was from internalised NMVs only. Data were analysed for mean fluorescence intensity using an LSRII flow cytometer using FACSDiva acquisition software.
5
Cytokine profiling in lung homogenates
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Lung homogenates were centrifuged, and the supernatants were collected. Filtered cell-free lung homogenates were used to detect IL-1α, IGF-1, IL-13, CCL3, CCL5, CXCL1, CXCL2 (R&D Systems), IL-2, TGF-β, IL-4, IL-5, IL-6, IL-12p40, IFN-γ, CCL2 (B&D Biosciences), IL-10, IL-17, IFN-β, TNF and GM-CSF (Biolegend) by enzyme-linked immunosorbent assay (ELISA).
6
Evaluating IL-12 Effects on HBV-Specific T Cells
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To study the effect of IL-12 on T cells, thawed PBMCs from two EPI patients were stimulated for 10 days with 2.5 µg/mL HBV core overlapping peptides (OLPs) (Genotype D, Proimmune, UK) in presence of 10 ng/ml rhIL-12 (Miltenyi Biotech). 41 HBV core OLPs were divided into 2 pools; HBV core peptides #1 including 20 peptides and HBV core peptides #2 including 21 peptides. Cultures were supplemented with 5 U/ml rhIL-2 (Invitrogen Life Technologies) at days 4 and 8. Cells were restimulated at day 10 with the same HBV core OLPs and rhIL-12 for 6 hours. 2 µg/ml Brefeldin A (Sigma-Aldrich) was added after 1 hour of restimulation. For cell surface staining, PBMCs were stained with CD3 (Biolegend), CD14 (BD Bioscience), CD19 (BD Bioscience), CD4 (BD Bioscience) and CD8 (Biolegend). Dead cells were excluded using the Aqua cell stain kit (Life technologies). After fixation and permeabilization of PBMCs with Foxp3/Transcription Factor Staining Buffer Set (Ebioscience/Thermofisher), they were stained for IFN-γ (BD Bioscience) and TNF (Biolegend) for 30 min. Samples were acquired using BD LSR Fortessa (BD Biosciences) and data were analysed in FlowJo version 9.9.4 (Treestar).
7
Murine Macrophage Cell Culture Protocol
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Murine macrophage cells (RAW 264.7) were obtained from the American Type Culture Collection (ATCC Accession number TIB-71). Dulbecco's modified Eagle's medium (DMEM), Minimum Essential Medium Eagle (α-MEM), and Fetal Bovine Serum (FBS) were purchased from Invitrogen-Gibco (Carlsbad, CA, USA). The mouse IL-3 (Cat number 432102, Lot: B154176), IL-4 (Cat number 431102, Lot: B156652), IL-6 (Cat number 431305, Lot: B165924), IL-1α (Cat number 433402, Lot: B171801), TNF (Cat number 430902, Lot: B170648), and enzyme-linked immunosorbent assay (ELISA) kit were purchased from BioLegend's ELISA MAX (San Diego, CA, USA). LPS derived from P. gingivalis (Pg) was purchased from Cedarlane (Ontario, Canada). For cells viability assay, alamarBlue was purchased from Invitrogen (Cat number DAL1025) (Grand Island, NY, USA) and protein concentrations were determined by Pierce BCA from Thermo Scientific (Cat number 23225) (Rockford, IL, USA). Mouse-derived RANKL, dimethyl sulfoxide (DMSO) ≥99.5%, penicillin G-streptomycin, acid phosphatase kits for tartrate-resistant acid phosphatase (TRAP) staining, 4,6-diamidino-2-phenylindole (DAPI) for DAPI staining, quercetin 98% (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one,3,3′,4′,5,6-entahydroxyflavone), and the other chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA).
8
Flow Cytometric Analysis of Murine and Human B Cells and Myeloid Subsets
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Flow cytometric analysis of murine B cells was performed by staining with fluorochrome-labeled antibodies against CD19, CD21 (both BD Biosciences), CD23, CD40, CD86 or IL-10 (all eBioscience) after fixation with 1.9% paraformaldehyde and permeabilization with 0.5% saponin (Sigma-Aldrich). Human B cells were stained for CD19, CD38 (both BD Biosciences), CD24, CD27 (both eBioscience), CD1d, IL-10, TNF (all Biolegend), and CD39 (Sony Biotechnology, San Jose, USA). Splenic myeloid cell subsets were discriminated using fluorochrome-labeled antibodies against CD11b, CD11c (both eBioscience), CD8, Ly6C (both Biolegend), F4/80 (AbD Serotec, Puchheim, Germany), Gr-1 (BD Biosciences), and Siglec-1 (Dr. J. den Haan, VUMC, Amsterdam, The Netherlands). Treg cells were fixed and permeabilized with the eBioscience Foxp3 fixation/permeabilization kit and stained using fluorochrome-labeled antibodies against CD3, CD4, Foxp3 (all eBioscience) and CD25 (BD Biosciences). All cells were stained with Aqua dye (Thermo Fisher Scientific) prior to fixation to discriminate dead cells. For all flow cytometric stainings, FcγR-binding inhibitor (2.4G2) was added and FMOs were used for gate setting. Flow cytometry was performed using a FACSCanto or Fortessa (BD Biosciences).
9
Cytokine Profiling of BMMC Activation
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2 × 105 BMMC were cultured in 0.25 mL of complete medium with or without VPA at 2 mM for 18 h. Cells were washed with HBSS and activated with different stimuli: L.m MOI 100:1 or Staphylococcus aureus Peptidoglycan (PGN; Sigma-Aldrich, USA) at 10 μg/mL or Pam3CSK4 (Pam; InvivoGen, USA) at 10 μg/mL, or Recombinant Listeriolysin-O (LLO; RayBiotech, USA) at 1000 ng/mL for 24 h. Supernatants were collected for the detection of TNF, IL-6, CCL2 (BioLegend, San Diego, CA., USA) and IL-13 (eBioscience, USA) by ELISA according to manufacturer’s instructions.
10
Quantification of Secreted Cytokines
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Mouse: Secreted CXCL12 (R&D systems), IFNλ (R&D systems), and IFNα (R&D systems) were measured by ELISA according to the manufacturer’s instructions. Human: Secreted CXCL12 (R&D Systems), IL-3 (R&D Systems), IL-6 (Biolegend), TNF (Biolegend), IFNλ (R&D systems), and IFNα (PBL assay science, Piscataway, NJ, USA) were measured by ELISA according to the manufacturer’s instructions.
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