The levels of TNF-α and IL-1β (Novus Biologicals, Inc.) in pleural effusions and the blood of mice were determined. Anti-CD8 antibody (Abcam), anti-PD1 antibody (EPR20665, Abcam), anti-PDL1 antibody (EPR20529, Abcam), anti-GAPDH antibody (ab8245, Abcam), and anti-β-actin antibody (13E5, CST) were used for antigen detection. Lymphocyte and tumor cell lines were co-cultured in 96-well plates at a ratio of 20:1. The LDH release experiment was then performed using the lactate dehydrogenase cytotoxicity test kit (Beyotime Biotechnology).
Tnf α
Manufactured by Novus Biologicals
Sourced in United States, China
TNF-α is a recombinant human tumor necrosis factor alpha protein. It is a cytokine involved in the regulation of a wide range of biological processes, including cell proliferation, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Proteintech (2 mentions)
Ifn γ, by Novus Biologicals (2 mentions)
Il 1β, by Novus Biologicals (1 mentions)
Epr20665, by Abcam (1 mentions)
Lactate dehydrogenase cytotoxicity test kit, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Halt protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp conjugate, by Jackson ImmunoResearch (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Goat anti mouse hrp conjugate, by Jackson ImmunoResearch (1 mentions)
Cytochrome c release assay kit, by GeneTex (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Wortmannin, by MedChemExpress (1 mentions)
Matrine, by MedChemExpress (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
D glucose solution, by Thermo Fisher Scientific (1 mentions)
Cd140b, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Abnova (1 mentions)
20 protocols using tnf α
1
Cytokine Levels and Lymphocyte Cytotoxicity
2
Western Blot Analysis of Apoptosis Markers
3
Establishing C2C12 Myotube Atrophy Models
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Three C2C12 myotube atrophy models were established, according to previous studies (31 (link)–33 (link)). Briefly, C2C12 myoblasts were differentiated to myotubes by culturing in 2% horse serum at 37°C. Dex (National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) or TNFα (Novus Biologicals, LLC, Littleton, CO, USA) were then added to the media for 48 h at 100 µM and 50 ng/ml, respectively. For the third model, the myotubes were incubated for 48 h in CM consisting of 33% ‘cachexia liquid’ and 66% fresh DMEM with 2% horse serum; this CM was replaced every 24 h. The ‘cachexia liquid’ was acquired as follows; when CT26 cells reached 90% confluence, their culture medium was replaced with 2% horse serum (differentiation medium) and the supernatant was collected as ‘cachexia liquid’ after 48 h. For the investigation of myoblast differentiation and myotube atrophy, matrine (Shanghai EFE Biological Technology Co., Ltd., Shanghai, China) was added to the culture medium at 0.1 and 0.2 mM for 48 h at 37°C. For the signalling pathway investigation, 0.1 mM matrine was added for 48 h at 37°C and 10 nM wortmannin (MedChemExpress, Monmouth Junction, NJ, USA) was added to culture medium for 48 h at 37°C.
4
Diabetic Pericyte-EC Interaction Modulation
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Diabetic media composed of EGM2 MV + 1% AGS supplemented with 30 mM D-glucose solution (Gibco, A2494001), 1 ng ml-1 TNF-α (Novus Biologicals, 210-TA-005/CF) and 1 ng m−1 IL-6 (Invitrogen, A42540) was applied from day 7 to day 14 or 28 to induce diabetic phenotypes. Diabetic media was newly prepared every 2 or 3 days and changed daily. EGM2 MV + 1% AGS supplemented with 30 mM D-Mannitol (Toronto Research Chemicals, TRC-M165000) in 1× PBS was used as an osmotic control, and EGM2 MV + 1% AGS only as an untreated control. Other treatments were prepared by supplementing EGM2 MV + 1% AGS with 10 μg ml−1 CD140b (PDGFRB) Monoclonal Antibody (APB5, PDGFRβ inhibitor, eBioscience, 14-1402-82), 10 µM Imatinib Mesylate (STI571, PDGFRβ inhibitor, ApexBio, A1805), 10 µM TIE2 kinase inhibitor (ApexBio, A5979), 10 µM DAPT (γ-secretase inhibitor, Tocris, 2634), 30 µM 19,20-dihydroxydocosapentaenoic acid (DHDP) or 500 µg ml-1 Advanced Glycation End product-BSA (AGE, Sigma-Aldrich, 121800-10MG). These treatments were applied from day 7 to 28 to alter pericyte-EC interactions, and fresh media were prepared every 2 days and changed daily.
5
Cytokine Expression Quantification in Mammary Tissue
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Polyclonal antibodies combined with the avidin-biotinperoxidase complex technique were used for the immunohistochemical detection of interleukin-1β (IL-1β) (Abnova, Walnut, CA, USA) and tumor necrosis factor-α (TNF-α) (Novus, SaintCharles, MO, USA). All samples from 1 animal were analyzed within the same assay run, and within each assay run treatment animals to be compared were included. The quantification of IL-1β and TNF-α protein expression level in mammary tissue samples was performed as previously described (Zhu et al., 2007 (link)). For each sample, a relative value of the amount of cytokine produced was expressed as the average percentage of the positively stained areas in 10 (for IL-1β) or 5 (for TNF-α) randomly selected view fields.
6
Regnase-3 regulates BMDM phagocytosis
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BMDMs from Regnase-3+/+ and Regnase-3−/− mice were differentiated as described in Regnase-3 protein expression in immune cell types, and 1.5 × 105 cells/well were plated in Macrophage-SFM on 96-well plates. BMDMs were left untreated or were preincubated for 4 h with 100 ng/ml LPS from S. enterica serotype typhimurium (Sigma-Aldrich), 1 µg/ml LTA from S. aureus (Sigma-Aldrich), 10 µg/ml high molecular weight poly-I:C (InvivoGen), or 50 ng/ml TNFα (Novus Biologicals). Cells were then probed with S. aureus bioparticles coupled to pH-sensitive pHrodo green fluorophore (Invitrogen) and centrifuged for 1 min at 200 g. The increase in fluorescence was acquired over time with a VarioSkan Lux microplate reader (Thermo Fisher Scientific).
7
Protein Expression Analysis Protocol
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The adherent tissue was collected and placed into a 1.5 mL centrifuge tube. After adding 200 UL of Ripa lysate, 1 UL of PMSF, 2 UL of phosphatase inhibitor, and 2 UL of protease inhibitor, they were placed in a tissue homogenizer. The tissue was homogenized for 2 min until it was fully broken and then placed on ice for 30 min. After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was put in a new 1.5 mL centrifuge tube and kept on ice. BCA protein concentration determination kit was used to determine the total protein concentration of different samples. The samples were electrophoresed through a 10 % SDS-PAGE gel and then transferred onto a PVDF membrane. After being blocked with 5 % skim milk powder, the membranes were incubated with antibodies against COX2, COL-I, COL-III (Bioss, China), TNF-α (Novus,USA), α-SMA (Abcam, USA), IL-1β (Proteintech,USA), and GAPDH (Bioss, China) at 4 °C overnight. After cleaning with Tris-buffered saline (TBS) 3 times, the secondary antibody was added and incubated for 2 h at room temperature. The membrane was washed three times with TBST (50 mL of Tris-HCl, 100 mL of NaCl, and 0.1 % Tween-20, pH 7.4) and scanned using the Odyssey Fc System (LICOR, USA). The densitometry of bands was measured by Image-Pro Plus 6.0 software (Media Cybernetics, USA).
8
Antibody-based Lipid Metabolism Assay
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Antibodies used in this study were as follows: Anti-FOXC2 (PA524588, Invitrogen), Anti-FA2H (15452-1-AP, Proteintech), Anti-GAPDH (2118, Cell Signaling Technology). Secondary antibodies included HRP Goat Anti-Mouse (926-80010, LI-COR) and HRP Goat Anti-Rabbit (926-80011, LI-COR). Cer(d18:0/24:0) and Cer(d18:0/24:1) were purchased from Avanti Lipids Polar. Fenretinide was purchased from MedChemExpress (HY-15373), and TNFα was purchased from Novus (#210-TA).
9
CD47 anti-apoptosis in cancer cells
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For CD47 anti-apoptosis function, cancer cells were treated with 20 ng/mL TNFα (Novus Biologicals, 210-TA/CF) or 20 ng/mL TNFα combined with 20 µg/mL SIRPα (R&D Systems, 9378-SA) for three days. For cell viability assay, 4000 cells were seeded in 96-well culture plates for treatments and lysed using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7571) at the end of the experiments. The luminescent signals were detected by Thermo Luminoskan Ascent Microplate Reader (Thermo Fisher Scientific). For apoptosis assay, cells were resuspended in Alexa Fluor 647 Annexin V (1:20, Biolegend, 640912) solution and incubated for 15 min at room temperature. Then, the Annexin V binding buffer (Biolegend, 422201) was added to the reaction, and the signals were analyzed by BD FACSCelesta Cell Analyzer (BD Biosciences) and FlowJo software.
10
Spheroids Response to Inflammatory Cytokines
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To investigate the response of spheroids to inflammatory conditions, they were treated with a cytokine cocktail containing TNF-α (biological activity > 1.0 × 107 U/mg; catalog no. NBP2-35185; Novus Biologicals LLC, CO, USA) and IFN-γ (biological activity 1 to 4 × 106 U/mg; catalog no. 575306; BioLegend, San Diego, CA, USA) at 10 and 20 ng/ml, respectively. At predetermined times, spheroids were retrieved, washed twice with PBS, and processed for qRT-PCR or flow cytometry.
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