1×104 BV2 cells were seeded in 24-well plates, and pretreated with RRx-001 at proportionate doses of 0.2, 1 and 5 μM and 5×104 primary microglial cells at doses of 0.2, 0.4 and 0.8 µM for 12 h, followed by LPS (100 ng/ml) for 24 h. After the cultured media were collected, the levels of TNF-α and IL-6 were measured by using a mouse TNF-α ELISA kit (Dakewe, Beijing, China) and mouse IL-6 ELISA kit (Boster Biological Technology co. ltd., Wuhan, China). Adhered cells were collected with trypsin and ultrasonicated to detect Nox4 activity with mouse NOX4 ELISA kit (Reddotbiotech, Kelowna, Canada). The absorbance was evaluated at 450 nm by using the infinite M1000 PRO multiscan spectrum (Tecan, Switzerland).
Mouse tnf α elisa kit
Manufactured by Dakewe
Sourced in China
The Mouse TNF-α ELISA kit is a laboratory equipment product designed to detect and quantify the levels of tumor necrosis factor-alpha (TNF-α) in mouse samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to provide a quantitative measurement of the target analyte.
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Lab products found in correlation
Mouse il 6 elisa kit, by Dakewe (4 mentions)
Mouse il 6 elisa kit, by BioLegend (2 mentions)
Mouse ifn γ elisa kit, by Dakewe (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Mouse il 6 elisa kit, by Boster Bio (1 mentions)
Infinite m1000 pro multiscan spectrum, by Tecan (1 mentions)
Anti mouse cd3 apc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd8a pe, by Thermo Fisher Scientific (1 mentions)
Mouse il 12p70 elisa kit, by Dakewe (1 mentions)
Mouse il 10 elisa kit, by Dakewe (1 mentions)
Anti mouse cd25 pe, by Thermo Fisher Scientific (1 mentions)
Apc anti mouse cd206, by Thermo Fisher Scientific (1 mentions)
Methylthiazoltetrazolium mtt, by Merck Group (1 mentions)
Il 6 elisa kit, by Dakewe (1 mentions)
Lipopolysaccharide lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Meilun (1 mentions)
Lps l2880, by Merck Group (1 mentions)
Atp tlrl atpl, by InvivoGen (1 mentions)
Mouse il 1β elisa kit, by Dakewe (1 mentions)
11 protocols using mouse tnf α elisa kit
1
Modulation of Neuroinflammatory Markers by RRx-001
2
Comprehensive Immune Profiling of Nanoparticle Formulations
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SF was provided by Shanghai Biochempartner Co., Ltd. (Shanghai, China). Injectable soya lecithin was provided by Shanghai Taiwan Pharmaceutical Co., Ltd. (Shanghai, China). Coumarin-6 (C6) was bought from Aladdin Chemical Co., Ltd. (Shanghai, RPC). DSPE-rhodamine B was purchased from Ruixi Biological Technology Co., Ltd (Xi’an, China). Methylthiazol tetrazolium (MTT) were purchased from Sigma-Aldrich (US). APC anti-mouse CD3, FITC anti-mouse CD4, PE anti-mouse CD8a, PE anti-mouse CD25, and Alexa Fluor® 647 anti-mouse FOXP3 were purchased from eBioscience. Alexa Fluor ® 488 anti-mouse CD86, PerCP/Cy5.5 anti-mouse F4/80, and APC anti-mouse CD206 were bought from eBioscience. Mouse IL-12p70 Elisa kit, Mouse TGF-β1 Elisa kit, Mouse IL-10 Elisa kit, and Mouse TNF-α ELISA kit were purchased from DAKEWE. All other reagents were of analytical grade and obtained commercially.
3
Cytokine Measurement in BALF and Cells
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After RT treated, mice lungs were irrigated three times with 1.0 mL PBS. The collected bronchoalveolar lavage uid (BALF) was immediately centrifuged to separate the cells and supernatant. Measurement of cytokine in the supernatants of BALF and RT-treated RAW264.7 cells was carried out using Mouse TNF-α ELISA kit (Dakewe, Shenzhen, China) and Mouse IL-6 ELISA kit (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions.
4
Innate Immune Response to Nucleic Acid Carriers
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For measuring the stimulation of the innate immune system, murine RAW264.7 macrophage cells were seeded on a 12-well plate and cultured in DMEM with 10% FBS. After 48 h, cells were treated with naked DNA, PDAPEI polyplexes and PEI polyplexes without serum for 4 h. Then, the treated cells were incubated in DMEM containing 10% FBS for 24 h. The cell-free supernatants were collected and analyzed using Mouse IL-6 ELISA kit (Dakewe Biotech; DKW12-2060-096; China) and Mouse TNF-α ELISA kit (Dakewe Biotech; DKW12-2060-096; China). The IL-6 and TNF-α level from the serum of Balb/c mice at 6 h after the subcutaneous injection of naked DNA, PDAPEI polyplexes and PEI polyplexes were measured using the same ELISA kit.
5
Inflammatory Cytokine Measurement in Mice
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Serum levels of inflammatory cytokines were measured using the mouse TNFα ELISA kit (Dakewe Biotech, 1217202) and IL-6 ELISA kit (Dakewe Biotech, 1210602) according to the manufacturer's protocols.
6
Inflammatory Signaling Pathway Modulation
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Lipopolysaccharide (LPS) from Escherichia coli O111:B4 was obtained from Sigma-Aldrich (Shanghai China). Dexamethasone sodium phosphate injection (DEX) was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd. GA (Cat#MB5206, meilunbio, China) was dissolved in dimethyl sulphoxide (DMSO) to obtain a stock concentration of 50 mg/kg and then diluted to the working concentrations in vitro experiments. In vivo experiments, GA was mixed with 0.9% normal saline (NS), and after ultrasound, a suspension was prepared for intragastric administration of mice. The rest of the supplies also include cell counting kit-8 (Cat#MA0218, meilunbio, China), mouse TNF-α ELISA kit (Cat#1217202, Dakewe, China), mouse IL-6 ELISA kit (Cat#1210602, Dakewe, China).
7
Inflammatory Cytokine Assay Protocol
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Dulbecco’s modified Eagle medium (DMEM, high glucose, 11965092), fetal bovine serum (FBS, 10099141), Lipofectamine™ 2000 (11668019), Pierce™ BCA Protein Assay Kit (23225), and Thermo Scientific RevertAid Mix (M16325) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Protease inhibitor cOmplete™ Cocktail (4693116001) was purchased from Roche (Basel, Switzerland). PerfectStart™ Green qPCR SuperMix (AQ601) was purchased from TransGen Biotech (Beijing, China). NucleoZOL (740404) was purchased from MACHEREY-NAGEL (MN, Düren, Deutschland). Mouse IL-1β ELISA kit (1210122), human IL-1β ELISA kit (1110122), mouse IL-6 ELISA kit (1210602), and mouse TNF-α ELISA kit (1217202) were purchased from DAKEWE (Shenzhen, China). LPS (L2880) was purchased from Sigma (Missouri, USA). ATP (tlrl-atpl) and MSU (tlrl-msu) were purchased from In vivoGen.
8
Evaluating Anti-inflammatory Effects of Compounds
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GPs were purchased from Jiatian Biotech Co., Ltd (Xi’an, China). The Dulbecco’s Modified Eagle’s Medium (DMEM) or RPMI 1640, fetal bovine serum (#10099-141, Gibco) and penicillin and streptomycin (#15070-063) were purchased from Thermo Fisher Scientific (Walthm, MA, USA). The mouse TNF-α ELISA kit (#1217202) and mouse IL-6 ELISA kit (#1210602) were purchased from Dakewe Biotech Co., Ltd. (Shenzhen, China). The TRizol reagent and cDNA synthesis kit were purchased from Takara (Kusatsu, Japan). The BCA Assay kit and the BeyoClick™ EdU-488 Cell Proliferation Kit (#C0071S) were purchased from Beyotime Biotech Co., Ltd (Shanghai, China). Antibodies of p-p65 (# 3033, 1:1000), p65 (# 8242, 1:1000), cleaved caspase-3 (#9661S, 1:1000), and caspase-3 Antibody (#9662S, 1:1000) were purchased from Cell Signaling Technology (Boston, MA, USA). Bax Antibody (#50599-2-Ig) and Bcl-2 Antibody (#12789-1-AP) were purchased from Proteintech (Chicago, IL, USA). Anti-GAPDH antibody (ab181602, 1:3000) was obtained from Abcam (Cambridge, UK). Ammonium pyrrolidinedithiocarbamate (PDTC, #P8765-1G) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The CCK8 assay kit (# C0038) was obtained from Dojindo Laboratories (Japan). The Annexin V-FITC Apoptosis Detection Kit (#556547) was purchased from BD Pharmingen™ (Franklin Lakes, NJ, US).
9
Cytotoxic T-cell Assay on GL261 Glioma
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GL261 cells were seeded in 96-well plates at 4000 cells per well in 0.1 ml medium. The next day, murine splenic lymphocytes were isolated using the Mouse Lymphocyte Separation Medium (7211011, Dakewe) according to the manufacturer’s instructions. Then the appropriate number of splenic lymphocytes was added in each well on top of the adhered GL261 cells. The ratio of splenic lymphocytes (effector cells) to GL261 cells in co-cultures was 20:1. Forty-eight hours post co-cultures, the production and secretion of IFN-γ or TNF-α in cell supernatants was measured with a mouse IFN-γ ELISA kit (1210002, Dakewe) or a mouse TNF-α ELISA kit (1217202, Dakewe), and the viability of the adhered GL261 cells was detected by MTT.
10
Evaluating Tumor-Immune Interactions
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4T1 cells were treated with PBS, HFn, Dox (2 µm ) or Dox@HFn (10 µm ) for 24 h. Then, 4T1 cells were co‐cultured with BMDC for 24 h. Subsequently, T cells and BMDC were cocultured at a 10:1 ratio for an additional 24 h. CD69 expression was detected by flow cytometry (BD Biosciences, USA). The supernatants obtained from the co‐culturing of T cells and DC for 24 h were used for cytokine analysis. The levels of IFN‐γ were measured using the Dakewe biotech Mouse IFN‐γ ELISA kit, following the manufacturer's instructions. Similarly, the levels of TNF‐α were determined using the Dakewe biotech Mouse TNF‐α ELISA kit, following the manufacturer's guidelines.
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