Osteoblasts were treated with 0.01 mg/mL cobalt-chromium-molybdenum (CoCr) particles with an average size of 0.5 µm, 0.05 µg/mL TNF (abcam®, UK) or with a combination of CoCr and TNF. Untreated cells served as negative controls, and cells stimulated with 1 µg/mL lipopolysaccharides (LPS; Sigma-Aldrich, Munich, Germany) and CoCr + LPS served as positive controls (Data shown in Supplementary Material
Tumor necrosis factor (tnf)
Manufactured by Abcam
Sourced in United Kingdom, United States
TNF is a cytokine that plays a central role in inflammation, immune system regulation, and cell signaling. 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
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Co2 incubator, by Thermo Fisher Scientific (3 mentions)
Z vad fmk, by Abcam (2 mentions)
Nupage, by Thermo Fisher Scientific (2 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Co2 atmosphere, by Thermo Fisher Scientific (1 mentions)
Mac 3, by BD (1 mentions)
Lsab2 kit, by Agilent Technologies (1 mentions)
Gm csf, by Abcam (1 mentions)
α sm actin, by Agilent Technologies (1 mentions)
Gm csfrα, by Santa Cruz Biotechnology (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Lamp1, by Abcam (1 mentions)
Anti mouse igg hrp, by GE Healthcare (1 mentions)
Hyperfilm ecl, by Cytiva (1 mentions)
Hybond p pvdf membrane, by Cytiva (1 mentions)
14 protocols using tumor necrosis factor (tnf)
1
CoCr and TNF Effects on Osteoblasts
2
Intravitreal Injection of TNF and Netarsudil
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Under anesthetization with intramuscular injections of a mixture of ketamine-xylazine, 10 ng TNF (Sigma-Aldrich, St. Louis, MO, USA) in 2 µL of phosphate-buffered saline (PBS) was administered intravitreally into the left eyes of the rats.19 (link) PBS was administered intravitreally into the contralateral right eye as a control. Netarsudil mesylate (Aerie Pharmaceuticals, Inc., Durham, NC, USA) was diluted in PBS and concurrent injection of 2, 20, or 200 pmol netarsudil and TNF was carried out intravitreally. The netarsudil alone administration group was also evaluated. A769662 (an AMPK activator, Abcam, Cambridge, UK) was diluted in dimethyl sulfoxide (DMSO) and simultaneous injection of 200 pmol A769662 and TNF was carried out intravitreally. The A769662 alone administration group was also evaluated. For this experiment, DMSO was used as a control. Dorsomorphin (an AMPK inhibitor; Abcam) was diluted in DMSO and pre-injected intravitreally 1 hour before second injection of 200 pmol netarsudil plus TNF. DMSO was also pre-injected intravitreally 1 hour before the second injection of DMSO, TNF, or 200 pmol netarsudil plus TNF.
3
Induction of Necrosis in Cell Lines
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HEK293T, HeLa, L929, HT-29, iBMDM, and MEF cells were grown in high-glucose-containing DMEM (Hyclone) supplemented with 10% FBS (Hyclone). TRIM25 KO HT-29 cells were generated using CRISPR/Cas9 technology. RIP3 KO HT-29 cells were kindly provided by Dr. Huayi Wang (ShanghaiTech University, Shanghai, China). All cells were grown at 37 °C under a humidified 5% CO2 atmosphere (Thermo Fisher). L929 cells were stimulated with 10 ng/mL TNF (Abcam) and 5 μM Z-VAD-FMK (Abcam) to induce cell necrosis, while other cells were induced necrosis with 100 nM Smac mimetic (MCE) addition.
4
Immunohistochemical Analysis of Lung Tissue
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Human and mouse sections from formaldehyde-fixed and paraffin-embedded lung tissues were deparaffinized and rehydrated. Epitope retrieval was performed by boiling the sections in citrate buffer, pH 6.0. Sections were reacted with hydrogen peroxide to block endogenous peroxidase, washed, and blocked with 5% goat serum. The sections were then incubated with the primary antibodies against GM-CSF (1:400; Abcam), TNF (1:400; Abcam), α-SM actin (1:1; Dako), CD31 (1:50; Dako), CD34 (1:1; Dako), CD68 (1:100; Dako), GM-CSFRα (1:50; Santa Cruz Biotechnology, Inc.), and Mac3 (1:200; BD) overnight at 4°C. After streptavidin-biotin amplification (LSAB2 kit [Dako] or Vectastain Elite ABC kit [Vector Laboratories]), the slides were incubated with 3, 3′-diaminobenzidine and counterstained with hematoxylin. A negative control was performed using mouse IgG or rabbit Ig instead of the primary antibody. The localization and intensity (0, 1+, 2+, 3+) of immunoreactivity were assessed by two independent examiners, blinded to the diagnosis of PAH or to the mouse treatment group. In the murine experiments described below, the number of Mac-3–positive cells per PA was counted for all vessels in the section and a mean value calculated per mouse.
5
ATDC5 Chondrocyte Inflammation Pretreatment
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The mouse chondrogenic cell line ATDC5 cells were seeded into Dulbecco's modified Eagle’s medium (DMEM, Gibco, USA) which was supplemented with 10% fetal bovine serum (Gibco, USA), penicillin (100 units/mL), and streptomycin (100 μg/mL). Following reaching confluency, the ATDC5 cells were pretreated for 24 hours with tumor necrosis factor ⍺ (TNF-⍺, Abcam, USA) (20 ng/mL) or phosphate buffer saline (PBS).
6
Western Blotting for TNF Quantification
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Total protein was extracted in 1% lysis buffer (RIPA, Merck Millipore, Hellerup, Denmark) containing a soluble protease inhibitor cocktail (Roche Diagnostics, Hvidovre, Denmark) according to Lambertsen et al. [16 (link)]. Protein concentrations were estimated using the Bradford Protein Quantification method.
Western blotting analysis for TNF (Abcam, Cambridge, UK, 1:2,000) was performed using 20 μg protein extract separated on bis/tris 4-12% SDS-PAGE gels (Nupage™, Invitrogen, Tåstrup, Denmark) essentially as previously described [16 (link)]. SeeBlue Plus2 pre-stained standard (Invitrogen) was used as a molecular weight marker and 0.5 ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included as a positive control. Densitometry was performed using Image J analysis software (version 1.47, National Institutes of Health (NIH), Bethesda, Maryland, USA) following recommendations of the Image J developers. Analysis was performed on two independent gels with two mice per group.
Western blotting analysis for TNF (Abcam, Cambridge, UK, 1:2,000) was performed using 20 μg protein extract separated on bis/tris 4-12% SDS-PAGE gels (Nupage™, Invitrogen, Tåstrup, Denmark) essentially as previously described [16 (link)]. SeeBlue Plus2 pre-stained standard (Invitrogen) was used as a molecular weight marker and 0.5 ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included as a positive control. Densitometry was performed using Image J analysis software (version 1.47, National Institutes of Health (NIH), Bethesda, Maryland, USA) following recommendations of the Image J developers. Analysis was performed on two independent gels with two mice per group.
7
Protein Expression Analysis by Western Blot
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Cells were lysed in Laemmli sample buffer containing β-mercaptoethanol (Sigma), protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Samples were run on SDS-PAGE gels and transferred onto Hybond-P PVDF membranes (Amersham, Buckinghamshire, UK). Membranes were blocked in 5% non-fat milk then probed with OPTN (Sigma), actin (Sigma), EEA1 (Cell Signaling, Hitchin, UK), LAMP1 (Abcam, Cambridge, UK), GM130 (BD, Oxford, UK), Golgin-245 (Santa Cruz, Heidelberg, Germany) or GAPDH (Santa Cruz) for MDM/THP-1 cells or OPTN (Abcam) and TNF (Abcam) for BMDMs overnight at 4°C and anti-rabbit IgG-HRP (Cell Signaling) or anti-mouse IgG-HRP (GE Healthcare, Buckinghamshire, UK) for 1 h at room temperature. Bound antibody was detected using ECL Plus (Amersham), exposed to Hyperfilm ECL (Amersham), quantified and normalised to actin using ImageJ (NIH).
8
Immunofluorescence Staining of Frozen Tissue Sections
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For immunofluorescence staining, primary antibodies were diluted in 1 % PBS-bovine serum albumin. Frozen sections were incubated with primary antibodies for agrin (R&D Systems, Minneapolis, MN; #AF550), Hu (Abcam, Cambridge, MA; #96474), CD45 (BioLegend, San Diego, CA; #30F11), anti-GFP (R&D Systems; #AF4240), CD11b (Bio-Rad, Hercules, CA; #M1/70.15), CSF1R (Invitrogen, Waltham, MA; #PA5-25974), F4/80(BM8) (Invitrogen; #41-4801-82), Iba1 (Invitrogen; #PA5-27436), NCAM (Invitrogen; #PA5-78402), Col4 (Abcam; #236640) and S100A1 (Invitrogen; #PA1-932), Ly6G (Abcam; #25377), MPO (R&D Systems; #AF3667), CD45 (BioLegend; #103102), G-SMA (SBC; #65638), TNF (Abcam; #183218), MMP2 (Invitrogen; #PA5-115583), and MMP10 (Invitrogen; #PA5-79677) overnight at 4°C, followed by secondary antibodies (Alexa Fluor 647, 546, and 488 conjugated anti-rabbit immunoglobulin G; Alexa Fluor 647 and 555 conjugated anti-goat immunoglobulin G; Alexa Fluor 488 and 594 conjugated anti-rat; Invitrogen) for 1 hour. Cell nuclei were visualized by DAPI. Sections were covered with aqueous Poly/Mount (Polyscience Inc) and examined with a Zeiss LSM 780 laser-scanning confocal microscope (Zeiss, Oberkochen) or Nikon Eclipse Ti2-E inverted microscope with an Abberior STED super resolution imaging platform. Images were compiled by ImageJ and Adobe Photoshop CS6 (San Diego, CA) software package.
9
Antibody Characterization for Integrin Studies
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The antibodies used in this study are LEAF™ purified Anti-mouse CD29 Armenian hamster IgG (Biolegend; HMB1-1), Armenian hamster IgG negative control (Biolegend), Itga6 (Serotec; NKI-GoH3; 1:200 in immunohistochemistry of frozen sections (IHC-Fr), cleaved Casp3 (Cell Signaling; 5A1E; 1:400 IHC-Fr), Ki67 (Abcam; ab66155; 1:300 IHC-Fr), CD45 (PTPRC) (BD Pharmingen; 30-F11; 1:250 in IHC-Fr, 1:200 in FACS), F4/80 (CiteAb; MF48020; 1:50 in IHC-Fr), Krt14 (Biosite; PRB-155P; 1:600 in IHC-Fr), Tnf (Abcam; ab6671; 1:200 in IHC-Fr), CD31 (PECAM1) (Biolegend; 1:50 in FACS), active Itgb1 (BD Pharmingen; 9EG7; 1:100 in IHC-Fr, 1:50 in FACS), total Itgb1 (CD29-Alexa Fluor 647) (Biolegend; HMβ1–1; 1:50 in FACS), CD49f (Itga6) (Biolegend; GoH3; 1:20 in FACS), SHARPIN (Proteintech; 1:1000 WB), Krt14 (Covance; 1:1000 WB), Vimentin (D21H3, Cell Signalling; 1:1000 WB), GAPDH (5G4 Mab 6C5, Hytest; 1:1000 WB), DyLight 680- or 800-conjugated IgGs (Thermo Scientific; 1:5000 WB) and AlexaFluor 488/568/647-conjugated secondary Igs (Invitrogen; 1:300 in IHC-Fr and FACS).
10
Western Blot Analysis of Inflammatory Signaling
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Western blot analysis for TNF (1:2,000, Abcam) was performed using 20 μg protein extract separated on 4–12% SDS-PAGE gels (Nupage, Invitrogen) essentially as previously described3 (link)4 (link). As a positive control, 0.5 ng 17 kDa murine recombinant TNF (Sigma Aldrich) was included. Western blotting analysis for SAPK/JNK (Cell Signaling, 1:1,000), phosphorylated (p)-SAPK/JNK (Thr183/Tyr185) (Cell Signaling, 1:1,000), p44/p42 MAPK (Cell Signaling, 1:1,000), p-p44/p-p42 MAPK (ERK1/2)(Cell Signaling, 1:1,000), p-38 (Cell Signaling, 1:1,000), p-p38 MAPK (Tyr180/Tyr182)(Cell Signaling, 1:1,000), and Iba1 (Wako, 1:500) was performed by resolving equal amounts of protein lysates by SDS-PAGE on Nupage Bis 4–12% gels, using MOPS SDS (Invitrogen) containing 0.25% antioxidant (Invitrogen) essentially as previously described6 (link)59 (link). TFIIB (1:1,000, Cell Signaling), GAPDH (1:2,500, Abcam) and α-actin (1:8,000, Millipore) were used as loading controls. SeeBlue Plus2 prestained standard (Invitrogen) was used as a molecular weight marker. Bands were quantified with Quantity One software (Biorad). Analysis was performed on 2–4 independent gels with n = 2–3/group and data were normalized to TFIIB, GAPDH or α-actin and represented as percentages relative to naïve TNFfl/fl mice or as ratios (for MAPK).
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