Forty healthy, clean mice weighing between 18 and 22 g were purchased from the Animal Center of Guangxi Medical University. The TNF-α ELISA kit was purchased from Biovision (product number K1052-100). JNK, TNF-α, p-JNK, Bcl-2, Bax, and β-actin antibodies were purchased from Yubo Biotechnology Co., Ltd. HRP goat anti-rabbit IgG was purchased from Boaosen Biotechnology. ECL color development kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd. BCA kits and protein extraction kits were purchased from Image Technology Co., Ltd. Trypsin, dimethyl sulfoxide, and fetal bovine serum were all purchased from Sigma (USA). DMEM was purchased from Beijing Kairuiji Biotechnology Co., Ltd.
Tnf α elisa kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The TNF-α ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay designed for the measurement of human tumor necrosis factor alpha (TNF-α) in various biological samples, such as cell culture supernatants, serum, and plasma.
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Lab products found in correlation
Il 1β elisa kit, by Abcam (2 mentions)
Potassium dihydrogen phosphate, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Pig iga elisa kit, by Abcam (1 mentions)
3 nitrotyrosine elisa kit, by Abcam (1 mentions)
Vcam1 elisa kit, by Thermo Fisher Scientific (1 mentions)
Nou39 k010, by Eagle Biosciences (1 mentions)
Mouse nt probnp elisa kit, by Elabscience (1 mentions)
Mouse il 1β elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 6 elisa kit, by Merck Group (1 mentions)
Il 6 elisa kit, by Abcam (1 mentions)
Human il 6 elisa kit, by Abcam (1 mentions)
Human interleukin il 1β elisa kit, by Abcam (1 mentions)
Lps escherichia coli o111 b4, by Merck Group (1 mentions)
P5726, by Merck Group (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
Mda elisa kit, by Abcam (1 mentions)
Trisodium citrate, by Merck Group (1 mentions)
33 protocols using tnf α elisa kit
1
Investigating TNF-α Signaling in Mice
2
Anti-inflammatory Activity Assay in RAW264.7 Cells
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Anti-inflammatory
activity was evaluated in the RAW264.7 cell line. The estimated TNF-α
content level after LPS exposure reflected the anti-inflammatory activity.
The TNF-α concentration of the cell culture supernatants was
determined using the TNF-α ELISA kit (BioVision, US). The experiment
was started after overnight culture in a 24-well plate (1 × 105 cells/well, 500 μL medium/well), the cells were pretreated
with the fucoidan extract (0.25–1 μg/mL) for 1 h and
1 μg/mL LPS for additional 24 h, and the culture supernatant
from each well was collected at the end of scheduled experiments and
used to measure TNF-α concentration. Quercetin, which is a strong
inhibitor for proinflammatory cytokine production, was used as the
positive control and a concentration of 50 μM was used for the
pretreatment before the LPS was treated on cells.
activity was evaluated in the RAW264.7 cell line. The estimated TNF-α
content level after LPS exposure reflected the anti-inflammatory activity.
The TNF-α concentration of the cell culture supernatants was
determined using the TNF-α ELISA kit (BioVision, US). The experiment
was started after overnight culture in a 24-well plate (1 × 105 cells/well, 500 μL medium/well), the cells were pretreated
with the fucoidan extract (0.25–1 μg/mL) for 1 h and
1 μg/mL LPS for additional 24 h, and the culture supernatant
from each well was collected at the end of scheduled experiments and
used to measure TNF-α concentration. Quercetin, which is a strong
inhibitor for proinflammatory cytokine production, was used as the
positive control and a concentration of 50 μM was used for the
pretreatment before the LPS was treated on cells.
3
Porcine Serum Cytokine and Immunoglobulin Analysis
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At the end of the experiment, blood samples were collected into 10‐mL tubes via jugular vein puncture and centrifuged at 3000 × g and 4 °C for 10 min to recover the serum using the Eppendorf® Centrifuge 5810/5810R analyzer (MilliporeSigma, Burlington, MA, USA) and blood sera were collected to analyze for serum cytokines [tumour necrosis factor (TNF)‐α, interleukin (IL)‐2] and immunoglobulins (Ig) (IgA, IgG and IgM) using porcine enzyme‐linked immunoabsorbent assay (ELISA) kits: TNF‐α ELISA Kit (ab100756), IL‐2 ELISA Kit (ABIN365284), Pig IgA ELISA Kit (ab190536), Pig IgG ELISA Kit (ABIN431274) and Pig IgA ELISA Kit (ab190536) in accordance with the manufacturer's instructions (Abcam, Cambridge, MA, USA). Fecal pH was measured using a portable pH meter. The three main SCFAs (acetate, butyrate and propionate) were analyzed in accordance with the methods described by Freire et al.19 Another 10 g of fresh fecal samples was collected on a pen basis, stored on an ice cooler and then transported to the laboratory for fecal microbial analysis.
4
Myocardial Oxidative and Inflammatory Markers
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Myocardial levels (n = 8–10 samples) of oxidative stress and inflammatory markers were tested using enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits: 3-nitrotyrosine ELISA kit (ab116691; Abcam), lipid peroxidation (malondialdehyde) assay kit (ab118970; Abcam), interleukin-6 (IL6) ELISA kit (ab100772; Abcam), intercellular cell adhesion molecule-1 (ICAM1) ELISA kit (ERICAM1; Thermo Fisher Scientific), vascular cell adhesion molecule-1 (VCAM1) ELISA kit (KHT0601; Thermo Fisher Scientific), and tumor necrosis factor alpha (TNFα) ELISA kit (ab108913; Abcam). Hydrogen peroxide (H2O2) was assessed in LV tissue homogenates (n = 8–10/group). Samples containing equal amounts of total protein were analyzed for H2O2 formation. H2O2 formation was measured by colorimetry at 540 nm. Results were converted using the standard curve for a known concentration of H2O2.
5
Plasma Biomarker Profiling in Mice
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ELISA analysis was performed according to the manufacturer’s instructions. The plasmic NE level in each group of mice was measured using an NE ELISA kit (NOU39-K010, Eagle Biosciences). The plasmic NT-proBNP level in mice was measured using a Mouse NT-proBNP ELISA Kit (E-EL-M0834c, Elabscience). The pro-inflammatory cytokines’ level in the cellular supernatant was measured by TNF-α ELISA Kit (ab208348, Abcam) and IL-1β Mouse ELISA Kit (BMS6002, Thermo).
6
LPS-induced Inflammation Assay in Mice
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The experimental procedures were approved (Approval number: 202003A-CNU-062) by the Animal Ethics Committee in Chungnam National University, and animal experiments were carried out per the approved protocols.
For animal experiments, all of the compounds were first pulverized with Tween 20 and then diluted with saline (1:99) to maximize the solubility of compounds in water [30 (link)]. The compounds in the saline/Tween 20 solution (2.5, 0.83 and 0.28 mg/mL, respectively) was administered via oral gavage (200 µL per injection). Mice (male BALB/C, 8 weeks; Samtako, Osan, Korea) were pretreated orally with individual compounds for 4 days. Two hours after the last treatment, the mice were injected i.p. with LPS (0.1 mg/kg) [31 (link)]. Ninety min after the LPS injection, the blood was collected, and the concentration of TNF-α in the blood was measured with TNF-α ELISA kit (Abcam, Cambridge, MA, USA). The experiment was performed with 3 mice per group.
For animal experiments, all of the compounds were first pulverized with Tween 20 and then diluted with saline (1:99) to maximize the solubility of compounds in water [30 (link)]. The compounds in the saline/Tween 20 solution (2.5, 0.83 and 0.28 mg/mL, respectively) was administered via oral gavage (200 µL per injection). Mice (male BALB/C, 8 weeks; Samtako, Osan, Korea) were pretreated orally with individual compounds for 4 days. Two hours after the last treatment, the mice were injected i.p. with LPS (0.1 mg/kg) [31 (link)]. Ninety min after the LPS injection, the blood was collected, and the concentration of TNF-α in the blood was measured with TNF-α ELISA kit (Abcam, Cambridge, MA, USA). The experiment was performed with 3 mice per group.
7
Serum Biomarker Profiling in Mice
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Mouse serum samples were collected periodically from the orbital plexus and analyzed by ELISA to determine their human AFP levels. Human AFP ELISA kits, mouse interleukin-6 (IL6) ELISA kit, and mouse tumor necrosis factor-α (TNFα) ELISA kit were purchased from Abcam. ELISA was performed according to the manufacturer’s instructions. The serum AFP, IL6, and TNFα levels were calculated according to each standard.
8
Quantification of Inflammatory Markers
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NCM460 cells were centrifuged at 5,000 × g at 4°C for 10 min, and then the supernatant was obtained. The concentrations of IL-6, TNF-α and NO in cell supernatant were detected using an IL-6 ELISA kit (RAB0306; Sigma-Aldrich), a TNF-α ELISA kit (ab181421; Abcam), and a nitric oxide ELISA kit (1227168631; klamar). The optical density value at 450 nm was assessed at the end of the experiment using a microplate reader.
9
Serum Immunoglobulin and Cytokine Analysis
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Serum IgG concentration was analyzed by IgG ELISA kit (ab283391, abcam, Cambridge, UK) according to kit’s manual. Serum IL-1β and TNF-α concentrations were determined by IL-1β ELISA kit (ab197742, abcam) and TNF-α ELISA kit (ab285327, abcam), respectively, according to the corresponding ELISA kit manual.
10
Inflammatory Cytokine Quantification
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The inflammation associated cytokines (TNF-α, IL-6 and IL-1β) in the experiments were detected by TNF-α ELISA Kit (#ab181421, Abcam, UK),IL-6 ELISA Kit (#ab46027, Abcam, UK) and IL-1β ELISA Kit (#ab46052, Abcam, UK) respectively. In brief, the culture supernants in the Control group and high-glucose treated group were collected. The expression levels of TNF-α, IL-6 and IL-1β were detected according to the manufacturer’s instruction.
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